Tape stripping methods for analysis of skin disease and pathological skin state

ABSTRACT

The present invention provides non-invasive methods for detecting, monitoring, and diagnosing skin disease and pathological skin states such as irritated skin and psoriasis. The methods include using tape stripping to analyze expression in epidermal samples, of one or more skin markers. In illustrative examples, the tape stripping is performed using pliable tape that has a rubber adhesive. Furthermore, the present invention provides methods for predicting and monitoring response to therapy for a skin disease, such as psoriasis or dermatitis. Finally, the methods can include the use of a microarray.

FIELD OF THE INVENTION

The invention relates generally to non-invasive diagnostics methods andmore specifically to methods for isolating and analyzing nucleic acidsfrom skin samples.

BACKGROUND OF THE INVENTION

Skin diseases represent major health care challenges today. For example,over five million Americans and over one hundred million peopleworldwide suffer from psoriasis. Detection, diagnosis, and staging of askin disease is an important aspect of its management. Currentdiagnostic methods rely mainly on visible observation and biopsies.However, detection methods for skin diseases that rely on visibleobservation are not effective for diagnosing many skin diseases, and donot detect diseases until after clinical manifestation. Invasive methodssuch as biopsies, not only are traumatic for a subject being tested,they also increase the risk of infection. Furthermore, invasive methodsdo not provide an enriched sample of cells on the surface of skin, whichare the cells involved in a surface reaction.

Detection and diagnosis of skin disease are important not only forpatient management, but also to assess the safety and efficacy of newskin disease therapeutic agents and new skin care products. Newtherapeutic agents are required for many skin diseases where presenttherapeutic agents are not fully effective. Furthermore, diagnosticmethods provide important information regarding the specific geneticchanges underlying a subject's skin disease. Identifying these geneticchanges identifies potential drug targets and may be critical indetermining whether a person will respond to a particular therapeuticagent.

In addition to assessing new therapeutic agents, detection and diagnosismethods are also important to assess the safety of new skin careproducts. Skin care products, including cosmetics, are an important partof most people's daily grooming habits. The average adult uses at leastseven different skin care products each day. Currently, all commercialskin care products are required to undergo safety testing. These teststake the form of Clinical Acute Primary Irritation and 14-day CumulativeIrritation Protocols followed by Human Repeat Insult Patch Testing(HRIPT) to detect sensitization (contact allergy). Visual analysis isused to determine the test results as described in Richard Berger andJames Bowman, A reappraisal of the 21-day cumulative irritancy test inman, J. Toxicol-Cut and Ocular Toxicol 1(2), 101–107 (1982). Therefore,allergic reactions are not detected until they have manifestedthemselves in a visible reaction.

In addition to issues related to relatively late stages of detection,visible analysis cannot distinguish subtle skin reactions that aredifficult to classify as irritant or allergic reactions. Thisclassification distinction is very important because it can be used asthe basis for deciding whether to continue to develop a new skin careproduct. Issues of irritation can be dealt with by reformulation,whereas issues of sensitization (i.e. an allergic reaction) can requiremore drastic product altering actions due to safety and liabilityconcerns. Therefore, misdiagnosis of irritant dermatitis as allergicdermatitis can block a safe and efficacious skin care product from beingavailable to people who could benefit from it.

SUMMARY OF THE INVENTION

The present invention is based on a non-invasive approach for recoveringnucleic acids such as DNA or messenger RNA from the surface of skin viaa simple tape stripping procedure that permits a direct quantitative andqualitative assessment of biomarkers. The method provides valuablegenetic information, not obtainable using a visible detection method.Furthermore, although tape-harvested RNA is shown to be comparable inquality and utility to RNA recovered by biopsy, the method providesinformation regarding cells of the outermost layers of the skin that isnot obtained using biopsy samples. Finally, the method is far lesstraumatic than a biopsy.

The method was applied to the analysis of gene expression duringirritant contact dermatitis. Using SLS irritation as a model system, theutility of assaying changes in IL-1β and IL-8 mRNA was tested as anindication of irritant skin reactions. It is show that both samplingmethods allow the recovery of RNA, the analysis of which revealscutaneous irritation. Data is presented that biopsy and tape-harvestedRNA are likely derived from different cell populations and that tapeharvesting is an efficient method for sampling the epidermis andidentifying select differentially regulated epidermal biomarkers.Furthermore, the Examples provided herein illustrate the successfulamplification of tape-harvested RNA for hybridization to DNAmicroarrays. These experiments show no significant gene expression leveldifferences between replicate sites on a subject and minimal differencesbetween a male and female subject. The array generated RNA profilesbetween normal and 24-hour 1% SLS-occluded skin were compared, and itwas observed that SLS treatment resulted in statistically significantchanges in the expression levels of more than 1,700 genes. These dataillustrate that tape harvesting as a non-invasive method for capturingRNA from human skin for microarray analysis.

Accordingly, provided herein is a method for characterizing skin of asubject, including applying an adhesive tape to a target area of skin ina manner sufficient to isolate an epidermal sample adhering to theadhesive tape, wherein the epidermal sample includes nucleic acidmolecules. At least one nucleic acid molecule whose expression isinformative of a skin disease or pathological skin state is detected inthe epidermal sample. The method of characterizing skin using tapestripping has a number of applications, such as the following: (i)disease classification/subclassification; (ii) monitoring diseaseseverity and progression; (iii) monitoring treatment efficacy; and (iv)prediction of the most beneficial treatment regimen. All of theseapplications, which themselves represent embodiments disclosed herein,rely on the concept of noninvasive sampling to recover information thatis otherwise difficult or impractical to recover (i.e. through the useof biopsies). This information is contained in the RNA of skin cellsclose to the surface of the skin. In one example, expression of one ormore of the genes listed in Table VII, or a combination thereof, isdetected in the epidermal sample to characterize the skin. Thisexemplary method is particularly useful for obtaining informationrelated to an irritated state of the skin.

Certain embodiments of the invention are based in part on the discoverythat in subjects afflicted with psoriasis, nucleic acid samples, forexample RNA samples, readily obtained from the epidermis of skin areasthat contain psoriatic lesions provide information regarding thedisease. Accordingly, the present invention provides a non-invasivemethod for isolating or detecting a nucleic acid molecule from anepidermal sample of a psoriatic lesion of a human subject. The methodincludes applying an adhesive tape to the psoriatic lesion of thesubject in a manner sufficient to isolate an epidermal sample adheringto the adhesive tape. The epidermal sample includes a nucleic acidmolecule that is then isolated and/or detected directly. The method oftape stripping psoriatic lesions can be used, for example, to monitorthe responsiveness of a psoriasis patient to treatment. Furthermore, themethod can be used to identify genes that are predictive of response totherapy.

Other embodiments are based in part on the discovery that for tapestripping of the skin, non-polar, pliable, adhesive tapes, especiallypliable tapes with rubber adhesive, are more effective than other typesof adhesive tapes. Using pliable tapes with rubber adhesives, as few as10 or less tape strippings and in certain examples as few as 4 or even 1tape stripping can be used to isolate and/or detect nucleic acidmolecules from the epidermal layer of the skin.

Accordingly, provided herein is a method for isolating and/or detectinga nucleic acid molecule from an epidermal sample from skin, includingapplying an adhesive tape to a target area of the skin in a mannersufficient to isolate an epidermal sample adhering to the adhesive tape,wherein the epidermal sample comprises nucleic acid molecules, andwherein the tape includes a rubber-based adhesive and is pliable.

In another embodiment, the present invention provides a method forquantitatively assessing gene expression of an amplified nucleic acid ina skin sample that overcomes prior difficulties in such a method.Accordingly, provided herein is a method for detecting a change in geneexpression, including applying an adhesive tape to a target area of skinand to an unaffected area of the skin, in a manner sufficient to isolatean epidermal sample adhering to the adhesive tape, wherein the epidermalsamples comprise nucleic acid molecules; and for each of the target areasample and the normal area sample, amplifying a target nucleic acidmolecule and a control nucleic acid molecule in the same experimentusing similar sample volumes and similar probes, wherein a change in therelative amplified levels of the target nucleic acid molecule to thecontrol nucleic acid molecule at the target area versus the normal areais indicative of a change in gene expression of the target nucleic acidmolecule at the target area.

In addition, provided herein is a method for detecting a response of asubject to treatment for psoriasis, including applying an adhesive tapeto the skin of the subject being treated for psoriasis, in a mannersufficient to isolate an epidermal sample, wherein the epidermal sampleincludes nucleic acid molecules; and detecting a target nucleic acidmolecule in the sample comprising nucleic acid molecules. Expression ofthe target nucleic acid molecule is informative regarding psoriasis.

In another embodiment the invention provides a kit for isolation anddetection of a nucleic acid from an epidermal sample, such as anepidermal sample from a psoriatic lesion or a target area of skinsuspected of being inflamed. The kit includes an adhesive tape forperforming methods provided herein. Accordingly, in one embodiment,provided herein is a kit that contains a pliable adhesive tape made upat least in part, of a non-polar polymer. In certain aspects, the tapeis a rubber-based tape.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 diagrammatically illustrates the experimental design of the SLSirritation protocol disclosed in further detail in Example 2.

FIGS. 2A, to 2C provide p-value distributions for gene expressionchanges upon induction of irritation by SLS exposure. The p-values,based on a regularized t-test distribution of all genes expressed atvalue above background in all replicate experiments grouped into 100bins and plotted against the number of genes in each bin. Panel A, the21,031 p-values of genes compared between untreated versus SLS occludedsamples. Panel B, the 21,307 p-values of genes compared between SLStreated versus water occluded samples. Panel C, 21,164 p-values of genescompared between untreated versus water occluded samples. The dashedlines in Panels A and B indicate the uniform distribution of p-valuesunder conditions of no differential expression.

DETAILED DESCRIPTION OF THE INVENTION

The specification hereby incorporates by reference in their entirety,the files contained on the compact discs filed herewith. Two copies ofthe compact disc are filed herewith. The compact disc includes a filecalled “6392999.xls,” created Mar. 31, 2004, which is 2.30 megabytes insize. Columns 1 and 2 of that spreadsheet file are identical to theTable included in the Appendix.

The present invention is based on a non-invasive approach for recoveringor analyzing nucleic acids such as DNA or RNA, from the surface of skinvia a simple tape stripping procedure that permits a direct quantitativeand qualitative assessment of pathologic and physiologic biomarkers.Tape-harvested RNA is shown to be comparable in quality and utility toRNA recovered by biopsy. The present method causes little or nodiscomfort to the patient. Therefore, it can be performed routinely in aphysician's office, for example, for point of care testing. Accordingly,provided herein are methods and markers for non-invasive isolationand/or detection of nucleic acids from epidermal samples using tapestripping.

The methods are effective for analysis of skin that is diseased or in apathological state, such as psoriatic skin or irritated skin. It isshown herein, that the methods can be used to characterize moleculardifferences in affected skin that visibly appears similar. Furthermore,it is shown herein that the method can be used to monitor response ofskin to treatment.

The methods can utilize a ΔC_(t) value, which provides usefulinformation regarding gene expression, especially in situations where itis difficult to obtain a “normal” nucleic acid sample, such as in tapestripping methods. Before the present disclosure it was taught that theΔC_(t) value was not being appropriate for gene expression analysis.

Methods of the present invention include a rapid, non-invasiveskin-sampling method for obtaining polynucleotides, including DNA andRNA. For example, mRNA is typically isolated in methods wherein geneexpression is analyzed. It is illustrated herein that improved isolationof nucleic acid molecules is obtained using pliable tape with a rubberadhesive.

Accordingly, in methods provided herein, an epidermal sample is obtainedby tape stripping the skin, which involves applying an adhesive tape tothe skin in a manner sufficient to isolate an epidermal sample adheringto the tape that includes nucleic acid molecules. Tape stripping methodsprovided herein, for example a single application of 4 individual tapes,do not result in glistening of uninvolved skin, and thus do not bare theviable epidermis. In contrast, a shave biopsy is expected to include notonly cells of the epidermis (primarily keratinocytes and melanocytes andimmune cells) but fibroblasts from the upper dermis as well.

A “biomolecule” is a specific binding pair member found in nature, orderived from a molecule found in nature. As used herein, the term“specific binding pair member” refers to a molecule that specificallybinds or selectively hybridizes to, or interacts with, another member ofa specific binding pair. Specific binding pair members include, forexample, analytes and biomolecules.

“Nucleic acid” means DNA, RNA, single-stranded, double-stranded ortriple stranded and any chemical modifications thereof. Virtually anymodification of the nucleic acid is contemplated. A “nucleic acid” canbe of almost any length, from 10, 20, 30, 40, 50, 60, 75, 100, 125, 150,175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000, 1500,2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000,10,000, 15,000, 20,000, 30,000, 40,000, 50,000, 75,000, 100,000,150,000, 200,000, 500,000, 1,000,000, 1,500,000, 2,000,000, 5,000,000 oreven more bases in length, up to a fill-length chromosomal DNA molecule.For methods that analyze expression of a gene, the nucleic acid isolatedfrom a sample is typically RNA.

The term “polypeptide” is used broadly herein to mean two or more aminoacids linked by a peptide bond. The term “fragment” or “proteolyticfragment” also is used herein to refer to a product that can be producedby a proteolytic reaction on a polypeptide, i.e., a peptide producedupon cleavage of a peptide bond in the polypeptide. A polypeptide of theinvention contains at least about six amino acids, usually containsabout ten amino acids, and can contain fifteen or more amino acids,particularly twenty or more amino acids. It should be recognized thatthe term “polypeptide” is not used herein to suggest a particular sizeor number of amino acids comprising the molecule, and that a peptide ofthe invention can contain up to several amino acid residues or more. Aprotein is a polypeptide that includes other chemical moieties otherthan amino acids, such as phosphate groups or carbohydrate moiety.

A “skin lesion” is a wound on the skin or injury to the skin. A ‘plaque”is a flattish, raised patch on the skin. “Scales” are thin flakes on theskin surface.

Throughout this application, the term “proliferative skin disorder”refers to a disease/disorder of the skin marked by unwanted or aberrantproliferation of cutaneous tissue. These conditions are typicallycharacterized by epidermal cell proliferation or incomplete celldifferentiation, and include, for example, X-linked ichthyosis,psoriasis, atopic dermatitis, allergic contact dermatitis, epidermolytichyperkeratosis, and seborrheic dermatitis. For example,epidermodysplasia is a form of faulty development of the epidermis.Another example is “epidermolysis”, which refers to a loosened state ofthe epidermis with,formation of blebs and bullae either spontaneously orat the site of trauma.

As used herein, the term “psoriasis” refers to a hyperproliferative skindisorder which alters the skin's regulatory mechanisms. In particular,lesions are formed which involve primary and secondary alterations inepidermal proliferation, inflammatory responses of the skin, and anexpression of regulatory molecules such as lymphokines and inflammatoryfactors. Psoriatic skin is morphologically characterized by an increasedturnover of epidermal cells, thickened epidermis, abnormalkeratinization, inflammatory cell infiltrates into the dermis layer andpolymorphonuclear leukocyte infiltration into the epidermis layerresulting in an increase in the basal cell cycle. Additionally,hyperkeratotic and parakeratotic cells are present.

The term “sample” refers to any preparation derived from skin of asubject. For example, a sample of cells obtained using the non-invasivemethod described above can be used to isolate polynucleotides,polypeptides, or lipids, preferably polynucleotides and polypeptides,most preferably nucleic acid molecules, such as polynucleotides, for themethods of the present invention. Samples for the present invention,typically are taken from a skin lesion, that is suspected of being theresult of a disease or a pathological state, such as psoriasis ordermatitis. The samples are taken of the skin surface of the suspiciouslesion using non-invasive skin sampling methods discussed herein.

The term “skin” refers to the outer protective covering of the body,consisting of the corium and the epidermis, and is understood to includesweat and sebaceous glands, as well as hair follicle structures.Throughout the present application, the adjective “cutaneous” can beused, and should be understood to refer generally to attributes of theskin, as appropriate to the context in which they are used. In apreferred embodiment, the skin is mammalian skin. In an illustrativeembodiment the skin is human skin.

The tape stripping methods provided herein typically involve applying anadhesive tape to the skin of a subject and removing the adhesive tapefrom the skin of the subject one or more times. In certain examples, theadhesive tape is applied to the skin and removed from the skin about oneto ten times. Alternatively, about ten adhesive tapes can be applied tothe skin and removed from the skin. These adhesive tapes are thencombined for further analysis. Accordingly, an adhesive tape can beapplied to and removed from a target site 10, 9, 8, 7, 6, 5, 4, 3, 2, or1 time, and/or 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 adhesive tape can beapplied to and removed from the target site. In one illustrativeexample, the adhesive tape is applied to the skin between about one andeight times, in another example, between one and five times, and inanother illustrative example the tape is applied and removed from theskin four times.

For the tape strippings, the same strip of tape can be repeatedlyapplied to, and removed from, a target site, such as a psoriatic lesion.However, in illustrative embodiments a fresh piece of adhesive tape issequentially applied to a target site of the skin. The individual tapestrips used to sample a site can then be combined into one extractionvessel for further processing such as nucleic acid extraction. In oneillustrative example, the adhesive tape is applied to the skin betweenabout one and eight times, in another example, between one and fivetimes, and in another illustrative example the tape is applied andremoved from the skin four times.

Factors such as the flexibility, softness, and composition of theadhesive tape used, the time the tape is allowed to adhere to the skinbefore it is removed, the force applied to the tape as it is applied tothe skin, the prevalence of a gene product being analyzed, the diseasestatus of the skin, and patient/patient variability are typically takeninto account in deciding on a protocol useful for a particular tapestripping method in order to assure that sufficient nucleic acids arepresent in the epidermal sample. A tape stripped sample includes, butmay not be limited to, tissues that are restricted to the surface ofskin and preferentially recovers vellus hair follicles and cells liningsebaceous, eccrine, and sweat ducts (i.e. the adnexal structuresassociated with the stratum corneum and epidermis), as well ascorneocytes. Tape stripping is stopped before viable epidermis isexposed by ceasing tape stripping before the tissue glistens. Therefore,the tape stripping method is considered a “noninvasive” method. Tapestripping sufficient to isolate an epidermal sample is tape strippingthat is performed on the skin sufficient times to obtain an RNA sample,wherein the tape stripping is stopped before the tissue glistens (i.e.becomes shiny, appears “moistened” or reflective).

Certain aspects of the invention, which themselves are embodiments ofthe invention, are based in part on the discovery that non-polar,pliable adhesive tapes, especially plastic-based adhesive tapes, aremore effective for obtaining nucleic acid samples from the skin thanother types of adhesive tapes. Using non-polar, pliable adhesive tapesas few as 10 or less tape strippings and in certain examples as few as 4or even 1 tape stripping can be used to obtain a nucleic acids that canbe analyzed. The method can be used as part of various embodimentsprovided herein, to isolate an RNA sample from the epidermis of skin,for gene expression analysis.

Accordingly, provided herein is a method of detecting expression ofgenes in the skin, including applying an adhesive tape to a target areaof the skin in a manner sufficient to isolate an epidermal sampleadhering to the adhesive tape, wherein the epidermal sample comprisesnucleic acid molecules, wherein the tape comprises a rubber adhesive,and wherein the tape is pliable. The nucleic acid molecules in theepidermal sample are then detected. The nucleic acid molecules, incertain examples, are applied to a microarray to detect the nucleic acidmolecules.

In another embodiment, provided herein is a method for isolating anucleic acid molecule from an epidermal sample from skin, includingapplying an adhesive tape to a target area of the skin in a mannersufficient to isolate an epidermal sample adhering to the adhesive tape,wherein the epidermal sample includes nucleic acid molecules, whereinthe tape includes a non-polar polymer adhesive, and wherein the tape ispliable. A nucleic acid molecule is then isolated from the epidermalsample. In illustrative examples, the non-polar polymer adhesive is arubber-based adhesive.

The rubber based adhesive can be, for example, a synthetic rubber-basedadhesive. The rubber based adhesive in illustrative examples, has highpeel, high shear, and high tack. For example, the rubber based adhesivecan have a peak force tack that is at least 25%, 50%, or 100% greaterthan the peak force tack of an acrylic-based tape such as D-squame™.D-squame™ has been found to have a peak force of 2 Newtons, wherein peakforce of the rubber based adhesive used for methods provided herein, canbe 4 Newtons or greater. Furthermore, the rubber based adhesive can haveadhesion that is greater than 2 times, 5 times, or 10 times that ofacrylic based tape. For example, D-squame™ has been found to haveadhesion of 0.0006 Newton meters, whereas the rubber based tape providedherein can have an adhesion of about 0.01 Newton meters using a textureanalyzer. Furthermore, in certain illustrative examples, the adhesiveused in the methods provided herein has higher peel, shear and tack thanother rubber adhesives, especially those used for medical applicationand Duct tape.

In addition to having higher peel, shear, and tack, the rubber-basedadhesive is more hydrophobic than acrylic adhesives. Furthermore, therubber based adhesive in illustrative examples is inert to biomoleculesand to chemicals used to isolate biomolecules, especially nucleic acidssuch as DNA and RNA. Finally, the adhesive can be relatively softcompared to other tapes such as D-squame™.

The rubber-based adhesive is on a support, typically a film, that makesthe tape pliable and flexible. In certain aspects, the tape can be softand pliable. “Pliable” tape is tape that is easily bent or shaped. “Softand pliable” tape is tape that is easily bent or shaped and yieldsreadily to pressure or weight. The film can be made of any of manypossible polymers, provided that the tape is pliable and can be usedwith a rubber adhesive. The thickness can be varied provided that thetape remains pliable. For example, the tape can be 0.5 mil to 10 mil inthickness, 1.0 to 5.0 mil in thickness. In one example, the tapecontains a rubber adhesive on a 3.0 mil polyurethane film. For examplethe film can be a polyurethane film such as skin harvesting tape(Product No. 90068) available from Adhesives Research, Inc (Glen Rock,Pa.).

The Examples illustrate tape stripping methods provided herein.Generally, before contacting a skin site with adhesive tape, a skin siteto be stripped is cleaned, for example using an antiseptic cleanser suchas alcohol. Next, tape is applied to a skin site with pressure. Pressurecan be applied for a fraction of a second, but can be applied forbetween 1 second and 5 minutes, typically between 10 seconds and 45seconds. In certain illustrative examples, tape is applied for 30seconds for each tape stripping. It will be understood that the amountof pressure applied to a skin site and the length of time for strippingcan be varied to identify ideal pressures and times for a particularapplication. Generally, pressure is applied by manually pressing downthe adhesive tape on the skin, however, objects, such as blunt, flatobjects can be used to assist in applying the tape to the skin,especially for areas of the skin from which it is more difficult toobtain nucleic acid samples from skin, such as uninvolved skin of asubject afflicted with psoriasis.

Virtually any size and/or shape of adhesive tape and target skin sitesize and shape can be used and analyzed, respectively, by the methods ofthe present invention. For example, adhesive tape can be fabricated intocircular discs of diameter between 10 millimeters and 100 millimeters,for example between 15 and 25 millimeters in diameter. The adhesive tapecan have a surface area of between about 50 mm² and 1000 mm², betweenabout 100 mm² to 500 mm² or about 250 mm².

As illustrated in the Examples provided herein, biopsy and tapestripping may not be equivalent sampling methods and therefore shouldnot be expected to yield identical results. Not intended to be limitedby theory, tape stripping, also referred to as “tape harvesting,” isrestricted to the skin surface and therefore may preferentially recovervellus hair follicles and cells lining sebaceous, eccrine and sweatducts as well as corneocytes (not predicted to contain RNA). Tapestripping methods provided herein, which typically utilize 10 or lesstape strippings, for example a single application of 4 individual tapes,do not result in glistening of uninvolved skin and thus do not bare theviable epidermis. Thus, tape stripping methods provided herein, providean epidermal sample. In contrast, a shave biopsy, in which a scalpelblade is use to slice a thin piece of skin from the surface (and whichtypically results in bleeding but does not require suturing), isexpected to include not only cells of the epidermis (primarilykeratinocytes and melanocytes and immune cells) but fibroblasts from theupper dermis. The potential enrichment of surface epidermis conveyed bytape stripping compared to a shave biopsy can be appreciated byconsidering that the surface area of a tape is 284 mm², while thesurface area of a 2×2 mm shave biopsy is 4 mm². Thus, tape-harvestedcells represent an enrichment of a sub-population of cells found in ashave biopsy. The data presented in Tables I and IV support thehypothesis that tape and biopsy-harvested RNA are derived from differentcell populations.

The epidermis of the human skin comprises several distinct layers ofskin tissue. The deepest layer is the stratum basalis layer, whichconsists of columnar cells. The overlying layer is the stratum spinosum,which is composed of polyhedral cells. Cells pushed up from the stratumspinosum are flattened and synthesize keratohyalin granules to form thestratum granulosum layer. As these cells move outward, they lose theirnuclei, and the keratohyalin granules fuse and mingle with tonofibrils.This forms a clear layer called the stratum lucidum. The cells of thestratum lucidum are closely packed. As the cells move up from thestratum lucidum, they become compressed into many layers of opaquesquamae. These cells are all flattened remnants of cells that havebecome completely filled with keratin and have lost all other internalstructure, including nuclei. These squamae constitute the outer layer ofthe epidermis, the stratum corneum. At the bottom of the stratumcorneum, the cells are closely compacted and adhere to each otherstrongly, but higher in the stratum they become loosely packed, andeventually flake away at the surface.

The skin sample obtained using the tape stripping method includes,epidermal cells including cells comprising adnexal structures. Incertain illustrative examples, the sample includes predominantlyepidermal cells, or even exclusively epidermal cells. The epidermisconsists predominantly of keratinocytes (>90%), which differentiate fromthe basal layer, moving outward through various layers having decreasinglevels of cellular organization, to become the cornified cells of thestratum corneum layer. Renewal of the epidermis occurs every 20–30 daysin uninvolved skin. Other cell types present in the epidermis includemelanocytes, Langerhans cells, and Merkel cells. As illustrated in theExamples herein, the tape stripping method of the present invention, isparticularly effective at isolating epidermal samples.

Nucleic acids can be isolated from the lysed cells and cellular materialby any number of means well known to those skilled in the art. Forexample, a number of commercial products available for isolatingpolynucleotides, including but not limited to, RNeasy™ (Qiagen,Valencia, Calif.) and TriReagent™ (Molecular Research Center, Inc,Cincinnati, Ohio) can be used. The isolated polynucleotides can then betested or assayed for particular nucleic acid sequences, including apolynucleotide encoding a cytokine. Methods of detecting a targetnucleic acid within a nucleic acid sample are well known in the art, andcan include microarray analysis, as discussed in more detail herein.

In another embodiment, provided herein is a non-invasive method foridentifying a predictive skin marker for response to treatment for adisease or pathological state, including: applying an adhesive tape tothe skin of a subject afflicted with the disease or pathological stateat a first time point, in a manner sufficient to isolate an epidermalsample including nucleic acid molecules and treating the subject for thedisease or pathological state. It is then determined whether the diseaseor pathological state has responded to the treatment, and if so, whetherexpression of a nucleic acid molecule in the epidermal sample ispredictive of response to treatment.

Expression of a nucleic acid molecule in the epidermal sample ispredictive of response to treatment if expression of the nucleic acidmolecule at the first time point is different in subjects that respondto treatment compared to subjects that do not respond to treatment. Itwill be understood that a variety of statistical analysis can beperformed to identify a statistically significant association betweenexpression of the nucleic acid molecule and response of the subject tothe treatment. For example, expression of the nucleic acid in certainexamples is elevated, in subjects that will not respond to treatment.Furthermore, expression of the nucleic acid can predict a level ofresponse to treatment, for example partial or temporary response totreatment versus a full response.

In another embodiment, provided herein is a non-invasive method forpredicting response to treatment for a disease or pathological state,including applying an adhesive tape to the skin of a subject afflictedwith the disease or pathological state in a manner sufficient to isolatean epidermal sample that includes nucleic acid molecules. A targetnucleic acid molecule is detected in the epidermal sample, whoseexpression is indicative of a response to treatment, thereby predictingresponse to treatment for the disease or pathological state.

The disease for embodiments directed at identifying a predictive skinmarker, or predicting response to treatment by detecting a predictiveskin marker, also referred to in these embodiments as a target nucleicacid molecule, can be virtually any skin disease. For example, the skindisease can be psoriasis or dermatitis, such as irritant contactdermatitis or allergic contact dermatitis. In aspects where the diseaseis psoriasis, the treatment can be, for example, a topical treatment,phototherapy, a systemic medication, or a biologic. Specific treatmentsare provided in Table VIII.

Samples from a tissue can be isolated by any number of means well knownin the art. Invasive methods for isolating a sample include the use ofneedles, for example during blood sampling, as well as biopsies ofvarious tissues. Due to the invasive nature of these techniques there isan increased risk of mortality and morbidity. The methods and kits ofthe present invention use a non-invasive sampling method to obtain askin sample. In certain examples, these methods are used along withconventional methods, such as a skin biopsy, to provide additionalinformation.

As mentioned above, tape-harvested cells appear to represent anenrichment of a sub-population of cells found in a shave biopsy.Accordingly, in certain aspects, in addition to a tape stripping methodprovided herein, a biopsy can be taken at the site of tape stripping,such as a psoriatic lesion site, or at another skin site. Nucleic acidmolecules from the biopsy can be isolated and analyzed. Analysis of thebiopsy data can be combined with analysis of data from a tape strippingmethod to provide additional information regarding the psoriatic lesion.

In certain aspects a nucleic acid molecule from uninvolved epidermaltissue is obtained by applying an adhesive tape to skin of the subjectin a manner sufficient to isolate an epidermal sample adhering to theadhesive tape, wherein the epidermal sample includes nucleic acidmolecules and wherein the skin is unaffected by a disease to be tested.Then a nucleic acid molecule is isolated and detected from the epidermalsample of the unaffected skin.

In certain aspects, the uninvolved skin can be from the upper arm or theupper back. These sites appear to provide relatively plentifulquantities of nucleic acid molecules using tape strippings. For example,tape stripping can be performed on uninvolved skin over the deltoid orupper back over the scapular spine and the periauricular region. Tapestripping generally involves the skin surface and therefore maypreferentially recover vellus hair follicles and cells lining sebaceous,eccrine and sweat ducts (i.e. adnexal structures) as well as corneocytes(not predicted to contain RNA).

Certain embodiments provided herein, are based in part on the discoverythat the expression of certain genes can be used to monitor response totherapy. Accordingly, in another embodiment, provided herein is a methodfor monitoring a response of a human subject to treatment for a diseaseor pathological state, including applying an adhesive tape to the skinof the subject being treated for the disease or pathological state at afirst time point and at least a second time point, in a mannersufficient to isolate an epidermal sample adhering to the adhesive tapeat the first time point and at the second time point. The epidermalsample includes a nucleic acid molecule, wherein a change in expressionof the nucleic acid molecule between the first time point and the secondtime point is indicative of a change in the disease or pathologicalstate.

In a related embodiment, provided herein is a method for detecting aresponse of a subject to treatment for a disease or pathological state,comprising: treating the subject for a skin disease or pathological skinstate; applying an adhesive tape to the skin of the subject in a mannersufficient to isolate an epidermal sample, wherein the epidermal sampleincludes nucleic acid molecules; and detecting a target nucleic acidmolecule in the sample comprising nucleic acid molecules. Expression ofthe target nucleic acid molecule is informative regarding the disease orpathological state. Therefore, the method identifies a response of thesubject to treatment for the disease or pathological state.

The detection can be a qualitative detection of whether the target geneis expressed, but is typically a quantitative expression leveldetermination. The method can be performed both prior to treatment andafter treatment. In one aspect, the method is performed after treatment,but before a change in disease or pathological state is observedvisually.

Time points for the monitoring and response-to-treatment methodsprovided herein, can include any interval of time, but are typically atleast 2 weeks, and more typically at least 1 month apart. For certainembodiments, time points are 2 months, 3 months, 6 months, 1 year, or 2years apart. Samples can be taken at any number of time points,including 2, 3, 4, 5, etc. time points. Comparison of expressionanalysis data from different time points can be performed using any ofthe known statistical methods for comparing data points to assessdifferences in the data, including time-based statistical methods suchas control charting. The disease or pathological state can be identifiedin the time series, for example, by comparing expression levels to acut-off value, or by comparing changes in expression levels to determinewhether they exceed a cut-off change value, such as a percent changecut-off value. In certain aspects, the first time point is prior totreatment, for example, prior to administration of a therapeutic agent,and the second time point is after treatment.

The disease or pathological state can be virtually any skin disorder.For example, the skin disorder can be psoriasis, dermatitis, or a skininfection, an allergic reaction, hives, seborrhea, irritant contactdermatitis, allergic contact dermatitis, hidradenitis suppurative,allergic purpura. Pityriasis rosea, Dermatitis herpetiformis, erythemanodosum, erythema multiforme, lupus erythematosus, a bruise, actinickeratoses, keloid, lipoma, a sebaceous cyst, a skin tag, xanthelasma,basal cell carcinoma, squamous cell carcinoma, or Kaposi's sarcoma. Incertain aspects, the disease or pathological state is other thanmelanoma.

The change in expression levels of at least one nucleic acid moleculecan be an increase or decrease in expression. Furthermore, depending onthe particular nucleic acid and the particular disease or pathologicalstate, an increase or decrease can indicate a response to treatment, ora lack of response. For example, the nucleic acid can encode a proteinsuch as CD2, TNFα, or IFNγ, and a decrease in expression at the secondtime point as compared to the first time point is indicative of positiveresponse to treatment for psoriasis. As another example, the method candetect a decrease in expression of TNFα, IFNγ, IL-12B, NPF, or IL-23B,wherein a decrease in expression is indicative of response to treatmentfor psoriasis. As another example, the method detects expression of akeratin 10, keratin 16, or keratin 17 gene product, wherein an increasein expression is indicative of response to treatment for dermatitis,such as irritant dermatitis.

In other aspects of this method, a population of genes are detected. Forthese aspects, the method can be performed using a microarray.

In another embodiment, provided herein is a method for characterizingskin of an animal subject, including applying an adhesive tape to atarget area of skin in a manner sufficient to isolate an epidermalsample adhering to the adhesive tape, wherein the epidermal sampleincludes nucleic acid molecules. A nucleic acid molecule whoseexpression is informative of a skin disease or pathological skin stateis then detected in the epidermal sample. For example, expression of agene listed in Table VII on the compact disk filed herewith can bedetected in the epidermal sample to characterize the skin for anirritated state. The Appendix included herewith, includes the list ofgenes found on Table VII.

The method of characterizing skin has a number of applications, such asthe following: (i) disease classification/subclassification; (ii)monitoring of disease severity and progression; (iii) monitoring oftreatment efficacy; and (iv) prediction of most beneficial treatmentregime. All of these applications, which themselves representembodiments disclosed herein, rely on the concept of noninvasivesampling to recover information that is otherwise difficult orimpractical to recover (i.e. through the use of biopsies). Thisinformation is contained in the RNA of skin cells close to the surfaceof the skin.

In one aspect of the method for characterizing skin, a test agent isapplied to the target area before the adhesive tape is applied.Accordingly, in one embodiment, provided herein is a method fordetermining the effect of an agent, such as a test agent, on skin,including: contacting a target area of the skin with the agent andapplying an adhesive tape to the target area of the skin in a mannersufficient to isolate an epidermal sample adhering to the adhesive tape,wherein the epidermal sample includes nucleic acid molecules. Nucleicacid molecules are optionally isolated from the epidermal sample todetermine an expression profile for the target site of the skin. Theexpression profile is indicative of a state of the skin, therebyproviding a determination of the effect of the agent on the skin. Theexpression profile can be obtained using a microarray, as discussed inmore detail herein.

A number of embodiments and aspects provided herein are directed attesting the effects of an agent, such as a test agent, on the skin. Inthese embodiments and aspects the agent can be applied until or beforeany visual symptoms become evident. For example, the agent can beapplied for between 1 second to 12 hours to a skin site, morespecifically the test agent can be applied between about 0.5 and 2 hoursbefore it is removed and tape stripping is performed on the skin sitecontacted with the agent.

In certain aspects, after exposure to a test agent, a test site isinterrogated by tape stripping and a molecular profile generated toclassify an agent. For example, the agent can be classified as highlyirritating or corrosive without damage to the skin. Furthermore, theagent can be classified as a specific type of irritant, for example adetergent or a dye.

The tape stripping according to these aspects of the invention isperformed to obtain an epidermal as disclosed in more detail herein. Forexample, to obtain the epidermal sample, an adhesive tape can be appliedand removed from the skin about one to ten times, as discussed in moredetail herein.

Methods performed herein for determining the effects of a test agent onskin can be performed as part of a process testing the safety and/orefficacy of the test agent. For example, the testing can be part oftesting performed as part of the approval process for marketing the testagent. As part of methods that analyze the effects of an agent, theagent is typically applied on the skin (i.e. topically). The agent canbe formulated as a paste, an ointment, a lotion, or a shake lotion, forexample. In certain aspects, the agent is a placebo.

In another aspect the invention provides a method of screening foragents or identifying agents that may cause skin disease or apathological skin state, or which may be used to treat skin disease or apathological skin state. In this aspect, for example, cells of the skin,such as epidermal cells, including keratinocytes and melanocytes, ordermal cells, such as fibroblasts, are contacted with a test agent. Theexpression of markers of the skin, disease or pathological skin state isthen detected.

The conditions under which contact is made are variable and will dependupon the type of agent, the type and amount of cells in the skin to betested, the concentration of the agent in the sample to be tested, aswell as the time of exposure to the agent. It will be understood thatroutine experimentation can be used to optimize conditions forcontacting skin with the agent.

An “agent” as used herein is used broadly herein to mean any molecule towhich skin is exposed. The term “test agent” or “test molecule” is usedbroadly herein to mean any agent that is being examined for an effect onskin in a method of the invention. For example, the agent can be abiomolecule or a small organic molecule. In illustrative examples, theagent is a peptide, polypeptide, or protein, a peptidomimetic, anoligosaccharide, a lipoprotein, a glycoprotein or glycolipid, achemical, including, for example, a small organic molecule, which can beformulated as a drug or other pharmaceutical agent, or a nucleic acid,such as a polynucleotide.

In certain aspects, the agent is a skin care product. Skin care productsare products designed to maintain healthy skin. They includeastringents, moisturizers, and sunscreens. Skin care products as usedherein, include personal care products, which are products that helpkeep skin and hair clean and fresh smelling include skin cleansers,shampoos, conditioners, and deodorants/antiperspirants. Furthermore,skin care products include cosmetics, which are skin care productsdesigned to color and adorn a surface of the body, such as the skin.Therefore, skin care products, as used herein, includes, for example,fragrances, astringents, moisturizers, sunscreens, skin cleansers, haircare items, deodorants/antiperspirants, colored cosmetics, haircosmetics, and nail cosmetics. Cosmeceuticals are skin care productsdesigned to go beyond strictly coloring and adorning the skin. Theseproducts actually improve the functioning of the skin and may be helpfulin preventing premature aging. Examples of these substances are alphahydroxy acids, such as glycolic acid, beta hydroxy acid, and salicylicacid. These hydroxy acids increase skin exfoliation making aging skinappear smoother and feel softer. Some vitamins, such as vitamin A(retinal), improve the appearance of aging skin by making the skinfunction better.

Skin care products can cause dermatitis in some individuals. It isimportant to distinguish dermatitis that is the result of irritated skinfrom dermatitis that is caused by an allergy, because allergic contactdermatitis is a more severe condition. Presently, methods are notavailable for distinguishing allergic contact dermatitis from irritantcontact dermatitis.

The Examples provided herein demonstrate using SLS irritation as a modelsystem, the utility of assaying changes in IL-1B and IL-8 mRNA has beentested as an indication of irritant skin reactions. The array generatedRNA profiles between normal and 24-hour 1% SLS-occluded skin werecompared, and it was observed that SLS treatment resulted instatistically significant changes in the expression levels of more than1,700 genes. These data not only identify markers of irritated skin, butalso illustrate that tape harvesting as a non-invasive method forcapturing RNA from human skin for microarray analysis.

Accordingly, provided herein is a method for characterizing skin of ananimal subject, including: applying an adhesive tape to a target area ofskin suspected of including irritated skin in a manner sufficient toisolate an epidermal sample adhering to the adhesive tape, wherein theepidermal sample includes nucleic acid molecules. A nucleic acidmolecule expressed from a gene listed in Table VII, which are identicalto the genes listed in the second column of the Table of the Appendix,or a combination thereof, is then detected in the epidermal sample,wherein expression of the nucleic acid molecule is altered in irritatedskin, thereby characterizing skin of the subject.

In another embodiment, provided herein is a method for diagnosing a skinrash as an infection by tape stripping the rash using methods disclosedherein. Nucleic acids isolated from the site of the rash can be analyzedfor the presence of nucleic acids of a microbe, wherein the presence ofnucleic acids of the microbe is indicative of an infection by themicrobe. The microbe can be, for example, a fungus, staphylococcus, orstreptococcus.

In another embodiment, provided herein is a method for distinguishing anirritant contact dermatitis (ICD) from an allergic contact dermatitis(ACD) in a subject, including: applying an adhesive tape to an area ofskin afflicted with dermatitis in a manner sufficient to isolate anepidermal sample adhering to the adhesive tape, wherein the epidermalsample comprises nucleic acid molecules. Then, determining expressionlevels of a gene associated with ICD or ACD, thereby distinguishing ICDwith ACD. Before expression levels are determined ribonucleic acid RNAmolecules can be optionally isolated from the epidermal sample.

In certain aspects, expression of more than one nucleic acid moleculecan be detected to characterize the skin, for example to distinguish ICDfrom ACD. As illustrated in the examples, expression of about 1700 genesis altered in irritated versus uninvolved skin. Therefore, changes ofskin state from normal to an irritated state, are accompanied by changesin at least 1700 genes. Therefore, in certain examples, methods providedherein characterize skin by analyzing expression of 2 or more, 5 ormore, 10 or more, 25 or more, 50 or more, 100 or more, 500 or more, 1000or more, 1500 or more, or all of the genes listed in Tables VII(Provided on the compact disk provided herewith as file 6392999.xls) orthe Table provided in the Appendix, which includes an identical list ofgenes as Table VII. In certain examples, expression is detected for agene listed in Table VI, which lists 100 genes identified in the studiesdisclosed herein, with the most dramatic expression changes in irritatedskin. For example, a detected nucleic acid can be an expression productof the IL-8 gene. In another example, the nucleic acid detected is thekeratin 10, 16 and/or 17 gene, in illustrative examples the keratin 16and/or 17 gene, wherein a down-regulation of the nucleic acid in a tapestripped skin is indicative of irritated skin.

The Examples provided herein illustrate the successful amplification oftape-harvested nucleic acids for hybridization to nucleic acidmicroarrays. These experiments show no significant gene expression leveldifferences between replicate sites on a subject and minimal differencesbetween a male and female subject.

Accordingly, in another embodiment, the present invention provides amethod for identifying an expression profile indicative of a disease orpathological state of a human subject, including applying an adhesivetape to an area of skin afflicted with the disease or pathological statein a manner sufficient to isolate an epidermal sample adhering to theadhesive tape, wherein the epidermal sample includes nucleic acidmolecules, and applying the nucleic acid molecules to a microarray.Nucleic acid molecules can optionally be isolated from the epidermalsample before being applied to the microarray. Expression levels of atleast 10 genes is then determined using the microarray; wherein analtered expression level for at least 2, 3, 4, 5, 6, 7, 8, 9, or each ofthe at least 10 genes as compared with expression in an epidermal samplefrom a normal sample identifies skin afflicted with the disease orpathological state, thereby identifying the expression profileindicative of the disease or pathological state.

In another embodiment, provided herein is a method for identifying anexpression profile indicative of a disease or pathological state of ahuman subject. The method includes applying an adhesive tape to an areaof skin afflicted with the disease or pathological state in a mannersufficient to isolate an epidermal sample adhering to the adhesive tape;and applying RNA molecules from the sample to a microarray to determinean expression pattern for the disease or pathological skin state sampleand the unaffected sample. A difference in the expression profile isindicative of an expression profile of a disease or pathological stateskin. The RNA molecules can optionally be isolated from the epidermalsample before being applied to the microarray.

In certain aspects, the disease or pathological state is dermatitis. Inother aspects, the disease or pathological state is psoriasis.

In embodiments where expression of more than 1 gene is analyzed, thedetection can be performed using a microarray. For example, themicroarray can include an array of probes, for example, directed to 2 ormore, 10 or more, 25 or more, 50 or more, 100 or more, 500 or more, 1000or more, 1500 or more, 1700 or more, or all of the genes listed in TableVII, or the subset of genes listed in Table VI. The microarrays formanother embodiment of the invention. Accordingly, in another embodiment,provided herein is a microarray that includes an array of probes, forexample, directed to 2 or more, 10 or more, 25 or more, 50 or more, 100or more, 500 or more, 1000 or more, 1500 or more, 1700 or more, or allof the genes listed in Table VII, or the subset of genes listed in TableVI.

For microarray expression analysis, approximately 0.1 to 1 milligram,typically 1 to 10 nanograms of RNA are isolated from an epidermalsample, for example obtained using a tape stripping method disclosedherein. Isolated RNA is then amplified and used for hybridization toprobes on a biochip. Amplification typically results in a total of atleast 1 microgram, and more typically at least 20 micrograms ofamplified nucleic acid. For example, amplification can be performedusing a MessageAMp™ a RNA kit (Ambion Inc.). Isolated RNA can be biotinlabeled before contacting the biochip such that binding to the targetarray can be detected using streptavidin. The probes bind specificallyto one or more of the genes listed in Tables VII and VIII, or acomplement thereof.

Hybridization of amplified nucleic acids to probes on a microarray istypically performed under stringent hybridization conditions. Conditionsfor hybridization reactions are well known in the art and are availablefrom microarray suppliers. For example, hybridization of a nucleic acidmolecule with probes found on a microarray can be performed undermoderately stringent or highly stringent physiological conditions, asare known in the art. For example, as illustrated in the Example sectionherein, hybridizations on a microarray can be carried out according tomanufacturer's (Affymetrix) instructions. For example, hybridization canbe carried out for 16 hours at 45° C. in a hybridization buffer composedof 100 mM MES, 1 M [Na+], 20 mM EDTA, 0.01% Tween 20. Washes can becarried out, for example, in a low stringency buffer ((6×SSPE, 0.01%Tween 20) at 25° C. followed by a high stringency buffer (100 mM MES,0.1M [Na+], 0.01% Tween 20) at 5° C. Another example of progressivelyhigher stringency conditions that can be used in the methods disclosedherein are as follows: 2×SSC/0.1% SDS at about room temperature(hybridization conditions); 0.2×SSC/0.1% SDS at about room temperature(low stringency conditions); 0.2×SSC/0.1% SDS at about 42° C. (moderatestringency conditions); and 0.1×SSC at about 68° C. (high stringencyconditions). Washing can be carried out using only one of theseconditions, for example, high stringency conditions, or each of theconditions can be used, for example, for 10 to 15 minutes each, in theorder listed above, repeating any or all of the steps listed.

As illustrated in the Examples provided herein, the manufacture and useof biochips such as those involving microarrays, also known asbioarrays, are known in the art. (For reviews of Biochips andmicroarrays see, e.g., Kallioniemi O. P., “Biochip technologies incancer research,” Ann Med, Mar; 33(2):142–7 (2001); and Rudert F.,“Genomics and proteomics tools for the clinic,” Curr Opin. Mol. Ther.,Dec; 2(6):633–42 (2000)) Furthermore, a number of biochips forexpression analysis are commercially available (See e.g., microarraysavailable from Sigma-Genosys (The Woodlands, Tex.); Affymetrix (SantaClara, Calif.), and Full Moon Biosystems (Sunnyvale, Calif.)).

Such microarrays can be analyzed using blotting techniques similar tothose discussed below for conventional techniques of detectingpolynucleotides and polypeptides, as illustrated in the Examplesprovided herein. Detailed protocols for hybridization conditions areavailable through manufacturers of microarrays. Other microfluidicdevices and methods for analyzing gene expression, especially those inwhich more than one gene can be analyzed simultaneously and thoseinvolving high-throughput technologies, can be used for the methods ofthe present invention. A microarray can provide for the detection andanalysis of at least 10, 20, 25, 50, 100, 200, 250, 500, 750, 1000,2500, 5000, 7500, 10,000, 12,500, 25,000, 50,0000, or 100,000 genes.

Quantitative measurement of expression levels using bioarrays is alsoknown in the art, and typically involve a modified version of atraditional method for measuring expression as described herein. Forexample, such quantitation can be performed by measuring a phosphorimage of a radioactive-labeled probe binding to a spot of a microarray,using a phospohor imager and imaging software.

Currently, primary irritation protocols are designed to detect highlyirritating and corrosive materials through limited patch testing.However, some of clinical sequelae associated with these protocols cantake days or weeks to manifest themselves. A testing protocol would befar superior if a substance could be applied for a shorter period oftime, for example 0.5–2 hours, and removed before any visual symptomsbecome evident. If a test site could be interrogated by tape strippingand a molecular profile generated that could classify an agent as highlyirritating or corrosive without damage to the skin, this would be anextremely useful and valuable test. Accordingly, presented herein is amethod for predicting subclinical irritant skin reactions, and the rapidprediction of irritant skin reactions before the manifestation ofclinical symptoms. These methods are useful to test the effects of anagent, such as a test agent on the skin.

Methods provided herein can be used to characterize the outer surface ofvirtually any animal. In certain aspects, the methods are used tocharacterize and/or otherwise analyze the outer surface of a body of amammalian subject. For example, the methods can be used to tape striprodents, such as mice, as well as, rabbits, or pigs. In illustrativeexamples, the methods are used to analyze human skin.

Certain embodiments of the invention relate specifically to psoriasisand are based in part on the discovery that nucleic acid samples, forexample RNA samples, from the epidermis of the skin can be obtained frompsoriatic lesions using tape stripping in psoriasis patients. Psoriasisis a chronic, genetic, noncontagious skin disorder that appears in manydifferent forms and can affect any part of the body, including the nailsand scalp. Psoriasis is categorized as mild, moderate, or severe,depending on the percentage of body surface involved and the impact onthe patient's quality of life (QoL). Psoriasis may be one of severaltypes: plaque psoriasis, pustular psoriasis, erythrodermic psoriasis,guttate psoriasis or inverse psoriasis. As indicated herein, methods forstaging provided herein, assist in determining the severity and type ofpsoriasis.

Although psoriasis may affect any area of the body, it is most commonlyfound on the scalp, elbows, knees, hands, feet, and genitals. Plaquepsoriasis, the most common type of the disease, is characterized byraised, thickened patches of red skin covered with silvery-white scales.Other types of psoriasis are characterized by different signs andsymptoms. For example, pustular psoriasis is characterized by pus-likeblisters, erythrodermic psoriasis is characterized by intense rednessand swelling of a large part of the skin surface, guttate psoriasis ischaracterized by small, drop-like lesions, and inverse psoriasis ischaracterized by smooth red lesions in the folds of the skin. Methodprovided herein help to distinguish between the various types ofpsoriasis.

Although psoriasis may be almost unnoticeable in its early stages,subjects often report an itching and/or burning sensation as the diseaseprogresses. In particular, plaque psoriasis usually begins with smallred bumps on the skin that progress to bigger, scaly patches that maybecome itchy and uncomfortable. As the scales accumulate, pink to deepred plaques with a white crust of silvery scales appear on the skinsurface.

The lesion suspected of being a psoriatic lesion can be the result ofKoebner's phenomenon. In this phenomenon psoriatic lesions appear at thesite of injury, infection or other skin problem. The lesion may mark theinitial onset of psoriasis, or may be a new lesion in an existing caseof psoriasis. In certain examples, the site of tape stripping can be onthe fingernails or toenails, which are known sites of psoriasis that canbe involved in psoriatic arthritis.

A “psoriasis skin marker” is a gene whose expression level is differentbetween skin samples at the site of a psoriatic lesion and skin samplesof uninvolved skin. Therefore, expression of a psoriasis skin marker isrelated to, or indicative of, psoriasis. As discussed herein, all of thepsoriasis skin markers illustrated herein exhibit increased expressionin psoriatic lesion versus non-psoriatic skin cell. Methods providedherein, for examples methods using microarrays to perform geneexpression analysis using samples obtained from tape stripped skin, canbe used to identify additional psoriasis markers. The expression ofthese psoriasis makers can increase or decrease in psoriatic lesions.

Many statistical techniques are known in the art, which can be used todetermine whether a statistically significant difference in expressionis observed at a 90% or preferably a 95% confidence level. In certainexamples, a greater than 4 fold increase or decrease can be used as acut-off value for identifying a psoriasis skin marker. The Examplesprovided herein illustrate the identification of psoriasis skin markers.In certain examples, there is at least a four-fold difference in levelsbetween a skin sample from a psoriatic lesion and non-lesional skin.Psoriasis skin markers identified herein include CD2, interferon γ(IFNγ), and tumor necrosis factor α (TNFα).

Accordingly, the present invention provides a non-invasive method forisolating or detecting a nucleic acid molecule from an epidermal sampleof a psoriatic lesion of a human subject, including applying an adhesivetape to the psoriatic lesion of the subject in a manner sufficient toisolate an epidermal sample adhering to the adhesive tape. The epidermalsample includes a nucleic acid molecule that is then isolated and/ordetected.

The nucleic acid for example can encode a protein such as CD2, TNFα. orIFNγ. Expression of these genes can be analyzed in psoriatic lesions.These embodiments, are useful for monitoring response to treatment forpsoriasis; for determining a treatment that is likely most effective,for genetically characterizing psoriasis; for diagnosing psoriasis; andfor identifying and analyzing nucleic acids that are predictive forresponse to a treatment for psoriasis. Changes in expression of thesegenes is shown in the Examples provided herein to be associated withpsoriasis. For example, expression of TNFα and CD2 are elevated in mostpatients with psoriasis. Furthermore, in certain patients TNFα, CD2, andIFNγ are elevated. Accordingly, in certain aspects, expression of TNFαand CD2 is analyzed. In other aspects, expression of TNFα, CD2, and IFNγare analyzed.

Methods of the present invention which isolate and detect a nucleic acidsample from an epidermal sample of a psoriatic lesion have utility notonly in detecting and staging a psoriatic lesion, but also indiagnosing, and prognosing psoriasis as well as monitoring response of apsoriatic lesion to treatment. These methods can also be used toidentify a predictive skin marker to identify a lesion and/or a patient,that will respond to treatment for psoriasis.

A biologic is a molecule derived from a living organism. Biologics usedto treat psoriasis typically target precise immune responses involvedwith psoriasis. Published data from studies suggests that pinpointingspecific immune responses produces less-toxic side effects because theentire immune system is not affected and neither are organs, such as theliver and kidneys. Some biologics work by either interfering with theabnormal T cells or blocking TNF-α. Typically, biologics must beinjected or infused to work.

The biologic can be, for example, alefacept or efalizumab, typicallyused for the treatment of adults who have moderate to severe chronicplaque psoriasis. Alefacept, which is typically given by intramuscularinjection once a week for 12 weeks, targets and kills a select group ofT cells that drive psoriasis.

Efalizumab, like alefacept, prevents T cells from becoming activated;and inhibits T cell trafficking. This prevents the T cells from enteringthe skin and causing inflammation. Efalizumab, which typically involvesweekly shots, unlike other systemic medications used to treat psoriasis,provides continuous therapy and is meant for long-term use.

Etanercept™, infliximab™ and adalimumab™ are biologic agents that blockTNF-α. In psoriasis, TNF-α is a chemical believed to be used by theimmune system that signals the skin to reproduce and mature too quickly(Gribetz, C. et al. “Clearing Psoriasis: A New Era of Optimism.”Contemporary Dermatology 2003: Vol. 1, No. 1: 1–8.). In certainillustrative examples, expression of a target gene believed to beinvolved in psoriasis is detected in a psoriatic lesion using a tapestripping method provided herein. If expression or elevated expressionis detected, a treatment can be administered to the subject that blocksa function of the target gene. For example, expression of TNFα can bedetected using the methods provided herein, and used to predict responseto biologics which target TNFα, such as etanercept™, infliximab™ andadalimumab™. For example, elevated levels of TNFα in an epidermal samplein skin can predict that a biologic that targets TNFα will be at leasttemporarily effective at treating psoriasis of the subject.

As illustrated herein, at least some psoriatic lesions express increasedlevels of TNFα. Therefore, methods herein to characterize a psoriaticlesion can be used to confirm that psoriatic lesions are expression TNFαbefore a subject is treated with a biologic such as Etanercept™,infliximab™ and adalimumab that block TNFα. Furthermore, as illustratedherein, IFNγ is not overexpressed in the psoriatic lesions of somepatients. Accordingly, in certain methods provided herein are used tocharacterize a psoriatic lesion for expression of IFNγ in order todetermine whether the subject is likely to respond to treatment with abiologic that targets abnormal T-cells. It is known that IFNγ isexpressed in T-cells.

TABLE VIII AAD Consensus Statement on Psoriasis Therapy (Callen, JP et.al. AAD Consensus statement on psoriasis therapies. J Amer Acad Dermatol2003: 49: 897–899) Topicals Phototherapy Systemic Plaque PsoriasisCorticosteroids UVB (with or w/o Methotrexate (many topicals and with orCyclosporin types/strengths) w/o oral retinoids) Retinoids TazarotenePhotochemotherapy (acitretin, Calcipotriene (with or w/o oraltazarotene*) Anthralin retinoids) Etanercept* Tar preparations AlefaceptKeratolytic agents Efalizumab (salicylic acid, Infliximab* lactic acid,urea) (Therapies may be combined Lubrication or used products withtopicals.) (Therapies may be combined or used in sequence shown.)Pustular Phototherapy Retinoids: (acitretin, isotretinoin*) MethotrexateCyclosporine Immunobio-logics (sometimes called “biologics”) (Therapiesmay be combined.) Guttae Topical therapies Phototherapy (with orSystemic w/o tar) therapies Photochemotherapy as needed ErythrodermicCyclosporine Methotrexate Retinoids (with or w/o phototherapy orphotochemotherapy (Therapies may be combined.) *Not yet approved by theU.S. Food and Drug Administration (FDA) for psoriasis.

Certain embodiments of the present invention are based in part on thediscovery that tape stripping psoriatic lesions can be used to monitorresponse of a subject to treatment for a skin disorder. For example,tape stripping can be used to monitor the response of one or morepsoriatic lesions, to treatment. The tape stripping methods can be usedto obtain a skin sample at a time point that is before a clinical changein a psoriatic lesion is observed or before a change in the severity ofpsoriasis is observable. Therefore, the tape stripping methods can beused to obtain information regarding whether a psoriasis patient isresponding to treatment before current methods can detect a response totreatment, or lack thereof.

The type and severity of psoriasis are usually measured visually. Forexample the severity of psoriasis can be measured using the NationalPsoriasis Grading Score (NPGS), which uses a variety of observablefactors, including redness, type of lesions, and amount of skin areaffected by redness. Methods provided herein provide an indication ofseverity and the type of psoriasis based on expression levels of genesassociated with psoriasis. These methods can be used to detect a changein psoriasis severity before these changes are observed visually, suchas using the NPGS. In certain aspects, the methods of the presentinvention are used in combination with a visual method, to determineresponse to treatment.

In another embodiment, provided herein is a method for characterizingpsoriasis in a subject including: analyzing expression of one or morenucleic acid molecules from an epidermal sample of a psoriatic lesion ofa subject. For this embodiment, typically the subject is a human subjectIn certain aspects, at least one of the nucleic acid molecules analyzedis a nucleic acid whose level of expression can effect choice oftreatment, such as TNFα, CD2 and/or IFNγ. Furthermore, it is illustratedherein that the tape stripping method can be successfully employed inexpression analysis using microarrays. Accordingly, microarray analysiscan be used to identify additional genes whose expression level isdifferent in psoriatic lesions of different patients, and whoseexpression level provides useful information regarding the type ofpsoriatic lesion, treatment choices, disease progression before clinicalsigns of change in disease, or the likelihood to respond to a therapy.

In another embodiment, the present invention provides a method fordiagnosing psoriasis in a human subject, including: applying an adhesivetape to a lesion suspected of being a psoriatic lesion on the skin ofthe subject in a manner sufficient to isolate an epidermal sampleadhering to the adhesive tape, wherein the epidermal sample includes atarget nucleic acid molecule. The target nucleic acid molecule is thedetected, wherein an altered expression of the target nucleic acidmolecule as compared with expression in an epidermal sample from asample not having psoriasis is indicative of psoriasis. In certainaspects, two or more target nucleic acid molecules are detected.

In certain aspects, two or more target nucleic acid molecules thatencode two or more proteins selected from CD2, TNFα, IFNγ; GAPDH,β-actin, IL-12B, IL-23A, Krt-16, Krt-17 are detected. In certainaspects, GAPDH and β-actin are used as controls, for example in ΔC_(t)calculations. In certain aspects a biopsy is taken at the site of theskin, and a nucleic acid sample is obtained from the biopsy. Forexample, in the biopsied sample expression of a target nucleic acidmolecule encoding a protein selected from TNFα, CD2, IFNγ, IL-12B,IL-23A, Krt-16, or Krt-17, is detected. Expression of all of these genesis known to be elevated in biopsied samples.

In the methods provided herein, tape stripping can be performed in aclinical setting by a first party that can send the tape strips to asecond party for nucleic acid detection. Nucleic acid isolation can beperformed by either the first party or the second party. For example,tape stripping can be performed in a physicians office by a nurse whosends the tape strips to a second party, such as an outside company whoperforms nucleic acid isolation and detection. Alternatively, nucleicacid isolation can be performed in the physicians office, who can sendthe isolated nucleic acid sample to a second party, such as an outsideservice provided, to perform nucleic acid detection and expressionanalysis.

Where two parties are involved in performing methods discussed herein,or where the methods disclosed herein are performed within the sameentity, revenue could be generated for performing the methods disclosedherein. For example, revenue can be generated for a service thatperforms a portion of the methods by accepting revenue in exchange fornucleic acid detection and expression analysis from tape strips.Furthermore, the service could generate an RNA profile and/or aclassification of the sample as ACD versus ICD or potentially corrosive.A corrosive substance can cause severe damage to the skin (e.g. sodiumhydroxide, 10% acetic acid). Therefore, provided herein is a method ofgenerating revenue by obtaining revenue for isolating and detecting anucleic acid in an epidermal sample obtained using tape stripping.Furthermore, revenue can be generated by selling kits, disclosed herein,that include adhesive tape for tape stripping skin, such asrubber-based, pliable adhesive tape. The kits could include RNAisolation reagents and optionally primers and probes for genes whoseexpression is correlated with a skin disease or pathological skin state.Furthermore the kit could include primers and probes for control genes,such as housekeeping genes. The primers and probes for control genes canbe used, for example, in ΔC_(t) calculations. The kits could alsoinclude instructions for performing tape strippings as well as foranalyzing gene expression using ΔC_(t) calculations.

In another embodiment, provided herein is a method wherein tapestripping is used to tape harvest skin sites in need of classification.Samples could be mailed to a laboratory of a service provider fordevelopment of an RNA profile which would indicate a classification(i.e. ACD versus ICD or corrosive potential) with greater than 95%confidence. The RNA profile could be available over an intranet orinternet for viewing by a customer of the service provider. In certainembodiments, a database is provided, of RNA profiles generated fromepidermal samples.

Skin samples obtained on adhesive films can be frozen before beinganalyzed using the methods of the present invention. Typically, this isperformed by snap-freezing a sample, as illustrated in the Examplesherein, using liquid nitrogen or dry ice.

One or more of the nucleic acid molecules in a sample provided herein,such as a as an epidermal sample, can be amplified before or after theyare isolated and/or detected. The term “amplified” refers to the processof making multiple copies of the nucleic acid from a single nucleic acidmolecule. The amplification of nucleic acid molecules can be carried outin vitro by biochemical processes known to those of skill in the art.The amplification agent can be any compound or system that will functionto accomplish the synthesis of primer extension products, includingenzymes. It will be recognized that various amplification methodologiescan be utilized to increase the copy number of a target nucleic acid inthe nucleic acid samples obtained using the methods provided herein,before and after detection. Suitable enzymes for this purpose include,for example, E. coli DNA polymerase I, Taq polymerase, Klenow fragmentof E. coli DNA polymerase I, T4 DNA polymerase, other available DNApolymerases, T4 or T7 RNA polymerase, polymerase muteins, reversetranscriptase, ligase, and other enzymes, including heat-stable enzymes(i.e., those enzymes that perform primer extension after being subjectedto temperatures sufficiently elevated to cause denaturation or thoseusing an RNA polymerase promoter to make a RNA from a DNA template, i.e.linearly amplified aRNA).

Suitable enzymes will facilitate incorporation of nucleotides in theproper manner to form the primer extension products that arecomplementary to each nucleotide strand. Generally, the synthesis willbe initiated at the 3′-end of each primer and proceed in the5′-direction along the template strand, until synthesis terminates,producing molecules of different lengths. There can be amplificationagents, however, that initiate synthesis at the 5′-end and proceed inthe other direction, using the same process as described above. In anyevent, the method of the invention is not to be limited to theamplification methods described herein since it will be understood thatvirtually any amplification method can be used.

One method of in vitro amplification, which can be used according tothis invention, is the polymerase chain reaction (PCR) described in U.S.Pat. Nos. 4,683,202 and 4,683,195. It will be understood that optimalconditions for a PCR reaction can be identified using known techniques.In one illustrative example, RNA is amplified using the MessageAmp™ aRNAkit (as disclosed in the Examples herein).

The primers for use in amplifying the polynucleotides of the inventioncan be prepared using any suitable method, such as conventionalphosphotriester and phosphodiester methods or automated embodimentsthereof so long as the primers are capable of hybridizing to thepolynucleotides of interest. One method for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066. The exact length of primer will depend on many factors,including temperature, buffer, and nucleotide composition. The primermust prime the synthesis of extension products in the presence of theinducing agent for amplification.

Primers used according to the method of the invention are complementaryto each strand of nucleotide sequence to be amplified. The term“complementary” means that the primers must hybridize with theirrespective strands under conditions, which allow the agent forpolymerization to function. In other words, the primers that arecomplementary to the flanking sequences hybridize with the flankingsequences and permit amplification of the nucleotide sequence. The 3′terminus of the primer that is extended can have perfect base pairedcomplementarity with the complementary flanking strand, or can hybridizeto the flanking sequences under high stringency conditions.

Upon isolation and optional amplification, expression of one or moregenes is analyzed. Analyzing expression includes any qualitative orquantitative method for detecting expression of a gene, many of whichare known in the art. Non-limiting methods for analyzing polynucleotidesand polypeptides are discussed below. The methods of analyzingexpression of the present invention can utilize a biochip, or otherminiature high-throughput technology, for detecting expression of two ormore genes.

The method of the present invention typically involve isolation of RNA,including messenger RNA (mRNA), from a skin sample. The RNA may besingle stranded or double stranded. Enzymes and conditions optimal forreverse transcribing the template to DNA well known in the art can beused. Alternatively, the RNA can be amplified to form amplified RNA. TheRNA can be subjected to RNAse protection assays. A DNA-RNA hybrid thatcontains one strand of each can also be used. A mixture ofpolynucleotides can also be employed, or the polynucleotides produced ina previous amplification reaction, using the same or different primerscan be so used. In certain examples, a nucleic acid to be analyzed isamplified after it is isolated. It is not necessary that the sequence tobe amplified be present initially in a pure form; it may be a minorfraction of a complex mixture.

In addition, RNAse protection assays can be used if RNA is thepolynucleotide to be detected in the method. In this procedure, alabeled antisense RNA probe is hybridized to the complementarypolynucleotide in the sample. The remaining unhybridized single-strandedprobe is degraded by ribonuclease treatment. The hybridized, doublestranded probe is protected from RNAse digestion. After an appropriatetime, the products of the digestion reaction are collected and analyzedon a gel (see for example Ausubel et al., CURRENT PROTOCOLS IN MOLECULARBIOLOGY, section 4.7.1 (1987)). As used herein, “RNA probe” refers to aribonucleotide capable of hybridizing to RNA in a sample of interest.Those skilled in the art will be able to identify and modify the RNAseprotection assay specific to the polynucleotide to be measured, forexample, probe specificity can be altered, hybridization temperatures,quantity of nucleic acid etc. Additionally, a number of commercial kitsare available, for example, RiboQuantTM Multi-Probe RNAse ProtectionAssay System (Pharmingen, Inc., San Diego, Calif.).

In another embodiment, a nucleic acid in the sample may be analyzed by ablotting procedure, typically a Northern blot procedure. For blottingprocedures polynucleotides are separated on a gel and then probed with acomplementary polynucleotide to the sequence of interest. For example,RNA is separated on a gel transferred to nitrocellulose and probed withcomplementary DNA to one of the genes disclosed herein. Thecomplementary probe may be labeled radioactively, chemically etc.

Detection of a nucleic acid can include size fractionating the nucleicacid. Methods of size fractionating nucleic acids are well known tothose of skill in the art, such as by gel electrophoresis, includingpolyacrylamide gel electrophoresis (PAGE). For example, the gel may be adenaturing 7 M or 8 M urea-polyacrylamide-formamide gel. Sizefractionating the nucleic acid may also be accomplished bychromatographic methods known to those of skill in the art.

The detection of nucleic acids can optionally be performed by usingradioactively labeled probes. Any radioactive label can be employedwhich provides an adequate signal. Other labels include ligands, coloreddyes, and fluorescent molecules, which can serve as a specific bindingpair member for a labeled ligand, and the like. The labeled preparationsare used to probe for a nucleic acid by the Southern or Northernhybridization techniques, for example. Nucleotides obtained from samplesare transferred to filters that bind polynucleotides. After exposure tothe labeled polynucleotide probe, which will hybridize to nucleotidefragments containing target nucleic acid sequences, the binding of theradioactive probe to target nucleic acid fragments is identified byautoradiography (see Genetic Engineering, 1 ed. Robert Williamson,Academic Press (1981), pp. 72–81). The particular hybridizationtechnique is not essential to the invention. Hybridization techniquesare well known or easily ascertained by one of ordinary skill in theart. As improvements are made in hybridization techniques, they canreadily be applied in the method of the invention.

Probes according to the present invention and used in a method of thepresent invention selectively hybridize to a target gene. In preferredembodiments, the probes are spotted on a bioarray using methods known inthe art. As used herein, the term “selective hybridization” or“selectively hybridize,” refers to hybridization under moderatelystringent or highly stringent conditions such that a nucleotide sequencepreferentially associates with a selected nucleotide sequence overunrelated nucleotide sequences to a large enough extent to be useful indetecting expression of a skin marker. It will be recognized that someamount of non-specific hybridization is unavoidable, but is acceptableprovide that hybridization to a target nucleotide sequence issufficiently selective such that it can be distinguished over thenon-specific cross-hybridization, for example, at least about 2-foldmore selective, generally at least about 3-fold more selective, usuallyat least about 5-fold more selective, and particularly at least about10-fold more selective, as determined, for example, by an amount oflabeled oligonucleotide that binds to target nucleic acid molecule ascompared to a nucleic acid molecule other than the target molecule,particularly a substantially similar (i.e., homologous) nucleic acidmolecule other than the target nucleic acid molecule.

Conditions that allow for selective hybridization can be determinedempirically, or can be estimated based, for example, on the relativeGC:AT content of the hybridizing oligonucleotide and the sequence towhich it is to hybridize, the length of the hybridizing oligonucleotide,and the number, if any, of mismatches between the oligonucleotide andsequence to which it is to hybridize (see, for example, Sambrook et al.,“Molecular Cloning: A laboratory manual (Cold Spring Harbor LaboratoryPress 1989)). An example of progressively higher stringency conditionsis as follows: 2×SSC/0.1% SDS at about room temperature (hybridizationconditions); 0.2×SSC/0.1% SDS at about room temperature (low stringencyconditions); 0.2×SSC/0.1% SDS at about 42EC (moderate stringencyconditions); and 0.1×SSC at about 68EC (high stringency conditions).Washing can be carried out using only one of these conditions, e.g.,high stringency conditions, or each of the conditions can be used, e.g.,for 10–15 minutes each, in the order listed above, repeating any or allof the steps listed. However, as mentioned above, optimal conditionswill vary, depending on the particular hybridization reaction involved,and can be determined empirically.

A method for detecting one or more genes can alternatively employ thedetection of a polypeptide product of one of these genes. For example,polypeptide products of one of the genes disclosed herein as associatedwith psoriasis or irritated skin, can be analyzed. The levels of suchgene products are indicative of psoriasis or a skin irritation whencompared to a normal or standard polypeptide profiles in a similartissue. Thus, the expression pattern of a gene disclosed herein asassociated with psoriasis or irritant dermatitis, will vary dependingupon the presence and stage of psoriasis or irritant dermatitisrespectively.

In this regard, the sample, as described herein, can be used as a sourceto isolate polypeptides. For example, following skin stripping, usingthe methods described above, cells isolated from the stratum corneum canbe lysed by any number of means, and polypeptides obtained from thecells. These polypeptides can then be quantified using methods known tothose of skill in the art, for example by protein microarrays, or ELISAanalysis.

In another embodiment, the present invention provides a method forobtaining gene expression data from amplified nucleic acids thatcompensates for variability in amplification reactions. In this method,relative expression of a target nucleic acid molecule and a controlnucleic acid molecule is compared to obtain relevant expression data.Accordingly, in certain embodiments provided herein, a ΔCt value isdetermined in order to identify gene expression changes. This value andmethod, although illustrated herein with respect to tape stripped skinsamples, can be used to identify differential gene expression in anytissue. It is especially useful, where it is relatively difficult toobtain sufficient RNA from a control sample.

The C_(t) values is the experimentally determined number ofamplification (e.g. PCR) cycles required to achieve a threshold signallevel (statistically significant increase in signal level (e.g.fluorescence) over background) for mRNA_(x) and a control mRNA (Gibson,Heid et al. 1996; Heid, Stevens et al. 1996). The Ct values aretypically determined using a target nucleic acid (e.g. mRNAx) primer andprobe set, and a control mRNA primer and probe set. A ΔC_(t) value iscalculated by calculating a difference in the number of amplificationcycles required to reach a threshold signal level between the targetnucleic acid molecule and the control nucleic acid molecule. Adifference in the ΔCt value at a target area versus another area of asubject's skin, such as a normal area, or an unaffected area, isindicative of a change in gene expression of the. target nucleic acidmolecule at the target area.

Using this value, altered expression is detected by comparing expressionof the target nucleic acid molecule with expression of a control nucleicacid molecule. The Examples provided herein, illustrate that the ΔC_(t)value, which is normally used to calculate a ΔΔC_(t) value (and thus acalibrated fold-change), is itself useful for characterizing thephysiologic state of the epidermis without reference to a calibrationsite. Example 2 provides the formula and related disclosure forcalculating a ΔC_(t) value. It is illustrated herein, that although theart teaches using a ΔΔC_(t) value and not a ΔC_(t) value for analyzingexpression data, a ΔC_(t) value is useful for this purpose, and providesthe advantage that it is not necessary to obtain a nucleic acid samplefrom a control site, where it may be difficult to obtain sufficientnucleic acid molecules.

The potential utility of ΔC_(t) values is illustrated in Example 2, bythe ΔC_(t, IL-8) for subject 4's SLS-treated skin (tape-harvestedsample; Table IV). The ΔC_(t) is −1.28, however it cannot be used tocalculate a ΔΔC_(t) value (and therefore a fold-change) becauseinsufficient RNA was recovered from the unaffected and water-occludedcontrol sites. However, comparison of this ΔC_(t) value to the remainingsubjects' average SLS ΔC_(t, IL-8) value of −0.89 and average valuesfrom tape-harvested water-occluded and uninvolved skin sites (>2.49 fornormal or 1.54 for tape) is highly suggestive that the ΔC_(t) value of−1.28 is in fact indicative of irritated skin. For example, the value of−1.28 implies that, compared to the average value for the 10 subjects,subject 4's SLS-site IL-8/β-actin mRNA ratio is at least2^(−(−1.28-2.49)) or 13.6-fold higher than the average value foruninvolved skin. A similar calculation using the average ΔC_(t, IL-8)for water-occluded samples as the calibrator suggests that theIL-8/actin mRNA ratio is 2^(−(−1.28-1.54)) or 7.1-fold elevated. Thesedata lead to the hypothesis that establishment of a database of ΔC_(t)values for different mRNA biomarkers might be useful to identify aphysiologic skin state without reference to an intrasubject controlsite. This utility of ΔC_(t) values is predicated upon the consistencyof the PCR reaction conditions and the use of identical probes betweensamples. Given these prerequisites, data in the Examples herein, supportthe potential for ΔC_(t) values being diagnostic indicators.

Accordingly, provided herein is a method for detecting a change in geneexpression, including: applying a first adhesive tape to a target areaof skin and a second adhesive tape to an unaffected area of the skin, ina manner sufficient to isolate an epidermal sample adhering to the firstadhesive tape and the second adhesive tape, wherein the epidermalsamples comprise nucleic acid molecules; and for each of the target areasample and the normal area sample, amplifying a target nucleic acidmolecule and a control nucleic acid molecule. For each of the targetarea sample and the normal area sample, a target nucleic acid moleculeand a control nucleic acid molecule are amplified and identifying, and aΔCt value by calculated by calculating a difference in the number ofamplification cycles required to reach a threshold signal level betweenthe target nucleic acid molecule and a control nucleic acid molecule,wherein a difference in the ΔCt value at the target area versus thenormal area is indicative of a change in gene expression of the targetnucleic acid molecule at the target area. The Ct values are typicallydetermined in the same amplification experiment (e.g. using separatereaction wells on the same multi-well reaction plate) using similarreaction conditions to other reactions.

The method for detecting a change in gene expression can be used alongwith the other embodiments provided herein to identify changes in geneexpression. For example, the method can be used to diagnose a skindisease or pathological skin state. In certain aspects, the method canbe used to detect a change in expression for any of the genes listed inTable VII, to assist in a characterization of a skin area as involvingirritant contact dermatitis.

Accordingly to the tape stripping method provided herein, a firstpopulation of adhesive tapes can be applied to the target region, and asecond population of adhesive tapes can be applied to a normal area ofskin or an unaffected area of skin. For example, four separate tapestrips can be applied to the target area of the skin and nucleic acidson the tape strips can be amplified together in a first reaction vessel.A different four separate tape strips can be applied to a normal area ofthe skin and nucleic acids on these tape strips can be amplifiedtogether in a second reaction vessel. In the first vessel and the secondvessel, both the control nucleic acid and the target nucleic acid arecan be amplified.

The target area for this embodiment, is typically an area of skinsuspected containing diseased skin or skin in a pathological state. Forexample, the target area can include a psoriatic lesion or a region ofskin with the characteristics of dermatitis.

In certain examples, the control nucleic acid molecule is expressed froma housekeeping gene. For example, the control nucleic aid molecule canencode β-actin, GAPDH, 18S rRNA, 28S rRNA, or tubulin. The adhesive tapeis typically applied one to ten times, or between one and ten identicaladhesive tapes are applied, as discussed herein related to the tapestripping method provided herein. Furthermore, a method according tothis embodiment can utilize a microarray to detect a population oftarget nucleic acid molecules.

In another embodiment, the present invention provides a method forsampling an epidermal layer other than skin, using the tape strippingmethod provided herein. For example, the tape stripping method can beused to obtain a nucleic acid sample from an epidermal layer of themouth, throat, or nose, or of an organ such as the liver, pancreas,kidney, intestines, stomach, bladder, brain, heart, or lungs, etc. byintroducing a tape strip into a subject and applying it to a surface ofthe organ. The organ can be sampled within a body of the subject orafter the organ is removed from the subject. Furthermore, the tapestripping method can be used to sample cells grown in vitro or organsreconstructed in vitro, for example for organ transplantation.

In another embodiment the invention provides a kit for isolation anddetection of a nucleic acid from an epidermal sample, such as anepidermal sample from a psoriatic lesion or a target area of skinsuspected of being inflamed. The kit can include an adhesive tape forperforming methods provided herein. Accordingly, in one embodiment,provided herein is a kit, including a pliable adhesive tape made up atleast in part, of a non-polar polymer. In certain aspects, the tapeincludes a rubber adhesive. In an illustrative example, the tape can beskin harvesting tape available (Product No. 90068) from AdhesivesResearch, Inc (Glen Rock, Pa.).

In addition to adhesive tape, the kit typically includes one or moredetection reagents, for example probes and/or primers for amplificationof, or hybridization to, a target nucleic acid sequence whose expressionis related to a skin disease or pathological state. The probes orprimers can be labeled with an enzymatic, florescent, or radionuclidelabel. For example, the probe can bind to a target nucleic acid moleculeencoding a protein selected from CD2, TNFα. IFNγ; GAPDH, or Krt-16.Alternatively, the probe can be, for example, an antibody that binds theencoded protein. The probes can be spotted on a microarray which isprovided in the kit.

The term “detectably labeled deoxyribonucleotide” refers to adeoxyribonucleotide that is associated with a detectable label fordetecting the deoxyribonucleotide. For example, the detectable label maybe a radiolabeled nucleotide or a small molecule covalently bound to thenucleotide where the small molecule is recognized by awell-characterized large molecule. Examples of these small molecules arebiotin, which is bound by avidin, and thyroxin, which is bound byanti-thyroxin antibody. Other labels are known to those of ordinaryskill in the art, including enzymatic, fluorescent compounds,chemiluminescent compounds, phosphorescent compounds, and bioluminescentcompounds.

The kit can include one or more primer pairs, including a forward primerthat selectively binds upstream of a gene whose expression is associatedwith psoriasis or irritant dermatitis, for example, on one strand, and areverse primer, that selectively binds upstream of a gene involved inpsoriasis or irritant dermatitis on a complementary strand. Primer pairsaccording to this aspect of the invention are typically useful foramplifying a polynucleotide that corresponds to a skin marker geneassociated with psoriasis or contact dermatitis using amplificationmethods described herein.

A kit provided herein can also include a carrier means beingcompartmentalized to receive in close confinement one or more containerssuch as vials, tubes, and the like, each of the containers comprisingone of the separate elements to be used in a method provided herein. Ifpresent, a second container may include, for example, a lysis buffer.The kit can alternatively include a computer-type chip on which thelysis of the cell will be achieved by means of an electric current.

Accordingly, kits provided herein can include an adhesive tape for tapestripping skin, such as rubber-based, pliable adhesive tape. The kitscould include RNA isolation reagents and optionally primers and probesfor genes whose expression is correlated with a skin disease orpathological skin state. Furthermore the kit could include primers andprobes for control genes, such as housekeeping genes. The primers andprobes for control genes can be used, for example, in ΔC_(t)calculations. The kits could also include instructions for performingtape strippings as well as for analyzing gene expression using ΔC_(t)calculations.

The present invention is not to be limited in scope by the specificexamples provided for below, which are intended. as single illustrationsof individual aspects of the invention and functionally equivalentmethods and components are within the scope of the invention.

EXAMPLE 1

Identification of Superior Tape Characteristics for Isolating NucleicAcid Acids from Skin Samples

The objective of this experiment was to compare adhesive films ofdiffering rigidity for the ability to remove epidermis and associatedtotal RNA from of surface of the skin.

The experimental protocol described here is designed to test thehypothesis that rigid tapes will remove more epidermis (and hencerecover more RNA) than an equivalent adhesive on a less rigid support.

TABLE 5 Product codes for adhesive tape test samples. Product PropertyCode Description Measured 413-201-1 3.0 mil clear PET (polyesterfilm)/413- Stiff backing 166-A (approx. 2.9 mil/2.0 mil ARMS) Adhesiveidentical to sample 413-92-1 413-92-1 3.0 mil PU (polyurethanefilm)/413-92-A Flexible backing (approx. 2.9 mil/2.0 mil ARMS releaseliner)

Procedure: Two main sites on the upper back with minimal hair werechosen for the tape stripping. Each site was about 40 mm×40 mm in sizeso as to allow three non-overlapping areas or “sub-sites” within themain site to be tape stripped. The main sites were in a similaranatomical location for all subjects. The main sites were cleansed withan alcohol pad and allowed to air dry 5 minutes. A test tape (obtainedfrom Adhesive Research, Glen Rock, Pa.) was applied with pressure to onesub-site and removed. This procedure was repeated 3 additional times (4total tape strips), each time with a fresh tape, to the same sub-site. Atotal of three sub-sites were tape-stripped. On each subject, one mainsite was tape stripped at the 3 sub-sites with tape 413-201-1; the othermain site was tape stripped at the 3 sub-sites with tape 413-92-1. RNAwas extracted from the tapes as described in Example 2 using a QiagenRNeasy™ kit and quantified by quantitative RT-PCR using the standardcurve method with total human spleen RNA as the standard.

Results and discussion: Table 6 shows the average mass of RNA recoveredper site for each subject. The data in Table 6 clearly shows that theadhesive with the flexible (i.e. pliable) backing (92-1) is superior inthe average amount of RNA recovered per site. Tape 92-1 collected on theaverage 6.1-fold more RNA than did tape 201-1.

TABLE 6 The average mass of total RNA recovered per site per subjectusing one adhesive formulation on two different backings. Average massrecovered per site¹ Mass ratio Subject Tape 201-1 Tape 92-1 92-1/201-1 10.86 6.09 7.09 2 1.23 4.48 3.64 3 0.37 2.95 7.97 4 0.50 4.64 9.31 5 2.0248.30 23.85 6 8.52 16.00 1.88 Average per site 2.25 13.74 6.11 acrosssubjects (±SEM) (±1.27) (±7.17) ¹The average mass of total RNA innanograms recovered per sub-site for each tape, in 6 subjects. RNA wasquantitated using quantitative RT-PCR by the standard curve method.

From the data in Table 6 it is concluded that the synthetic rubberadhesive on a pliable polyurethane film is superior for the purposes ofRNA recovery than the same adhesive on a stiff polyethylene filmbacking.

Next, a synthetic rubber adhesive formulation was compared to an acrylicadhesive formulation for the ability to retrieve RNA from epidermalcells recovered from the surface of uninvolved skin and water-occludedskin by tape stripping.

TABLE 7 Descriptions of adhesive tape films used in this protocol.Adhesive ID Description 413-92-1 3.0 mil polyurethane film; 413-92-A(approx. 2.9 mil/2.0 mil ARMS release liner); rubber-based adhesive(Product 90068) 413-92-3 3.0 mil polyurethane film; 413-92-C (approx.2.8 mil/2.0 mil ARMS release liner); acrylic-based adhesive

Site selection and harvesting procedure: The back was chosen as the sitefor this protocol. Sites were cleansed with alcohol and allowed to airdry before applying the water soaked patch. The water patches contained0.25 ml of distilled, sterile water. Patches remained attached for24-hours.

Subjects returned to the clinic 24-hours later for patch removal. Oncethe patches were removed, the patched sites were not cleansed, butsimply allowed to air dry for 15 minutes; the uninvolved skin site wascleansed with an alcohol patch and allowed to air dry for 5 minutes.After air-drying, and visual grading, 3 water and 3 adjacent uninvolvedskin sites were tape-stripped with tape 413-92-1 four times each (4 newtapes per site). The remaining 3 water and 3 uninvolved skin sites weresimilarly harvested with tape 413-92-3.

Results and discussion: In this experiment two different adhesiveformulations were tested for the ability to recover RNA by tapeharvesting the surface of the skin.

TABLE 8 Average mass of total RNA recovered per site from normal andwater treated skin in 7 subjects. Average RNA Yield by Tape and SkinTreatment Tape 92-1 (rubber) Tape 92-3 (acrylic) Uninvolved WaterUninvolved Water Subject skin¹ Occluded¹ skin¹ Occluded¹ 1 3.6 0.770.076 0.16 2 62 14.9 ND ND 3 6.3 3.2 0.069 ND 4 0.54 0.14 ND ND 5 12.18.5 0.026*  0.78* 6 5.9 10 0.50* ND 7 1.2 2.1 1.06 1.11 Subject 13.1 ±8.3 5.7 ± 2.1 0.35 ± 0.2 0.68 ± 0.28 average ± SEM ¹The average mass oftotal RNA recovered per site is shown in nanograms; unless otherwisenoted, each value is the average of 3 sub-sites; ND indicates that noRNA was detected; *indicates that the value is an average of less than 3sites because RNA was not detected at all sites

Table 8 clearly reveals that tape 92-1 is superior at recovering RNAfrom normal and water-occluded skin. In conclusion, a prototype rubberadhesive film (Product 90068) (Adhesive Research, Inc., Glen Rock, Pa.)was tested against an acrylic adhesive for the ability to recover RNAfrom, the surface of the skin. The results conclusively demonstrate thatthe prototype rubber-based film is better than the acrylic film atrecovering RNA from normal and water-occluded skin sites.

EXAMPLE 2

The Use of RT-PCR and DNA Microarrays to Characterize RNA Recovered byNon-Invasive Tape-Harvesting of Normal and Inflamed Skin

This example illustrates a non-invasive approach for recoveringmessenger RNA from the surface of skin via a simple tape strippingprocedure that permits a direct quantitative and qualitative assessmentof pathologic and physiologic biomarkers. Tape-harvested RNA is shown tobe comparable in quality and utility to RNA recovered by biopsy. UsingSLS irritation as a model system, the utility of assaying changes inIL-1β and IL-8 mRNA has been tested as an indication of irritant skinreactions and show that both sampling methods allow the recovery of RNA,the analysis of which reveals cutaneous irritation. Data is presentedthat biopsy and tape-harvested RNA are likely derived from differentcell populations and that tape harvesting is an efficient method forsampling the epidermis and identifying select differentially regulatedepidermal biomarkers. The successful amplification of tape-harvested RNAis reported, for hybridization to DNA microarrays. These experimentsshowed no significant gene expression level differences betweenreplicate sites on a subject and minimal differences between a male andfemale subject. The array generated RNA profiles was also comparedbetween normal and 24-hour 1% SLS-occluded skin and observed that SLStreatment resulted in statistically significant changes in theexpression levels of more than 1,700 genes. These data establish theutility of tape harvesting as a non-invasive method for capturing RNAfrom human skin.

Contact dermatitis, a common skin reaction, involves several signalingpathways. Irritant contact dermatitis (ICD) predominantly involveskeratinocyte activation (Freedberg, Tomic-Canic et al. 2001), whereasLangerhans cell presentation of antigen to T-cells in draining lymphnodes and recognition of the offending allergen in skin by memoryT-cells control the initiation and expression of allergic contactdermatitis (ACD; (Feghali and Wright 1997)) Clinically, both contactdermatitides are characterized by pruritus, erythema and edema. Thiscommonality of the clinical signs and symptoms makes distinguishingbetween ICD and ACD difficult at the clinical level, particularly whensymptoms are subtle. By contrast, at the molecular level, ICD and ACDare believed to be characterized by unique mRNA patterns, although thepublished literature is conflicting (Hoefakker, Caubo et al. 1995;Flier, Boorsma et al. 1999; Morhenn, Chang et al. 1999; Ryan andGerberick 1999; Ulfgren, Klareskog et al. 2000; Cumberbatch, Dearman etal. 2002). Documentation of simple and complex mRNA profiles is possibleusing reverse transcriptase polymerase chain reaction (RT-PCR) and DNAmicroarray technologies. Using the technique of tape stripping, RNA canbe harvested from both normal and inflamed skin and by combining tapestripping and RNA profiling, it may be possible to non-invasivelyestablish a diagnosis of ICD or ACD.

The study disclosed in this Example demonstrates that sufficient RNA canbe recovered using sequential application of as few as 4 small tapes toa skin site. In order to document the use of tape harvesting as anaccurate and reliable sampling method we performed a clinical trial inwhich occlusive patches containing either 1% SLS (irritant) or water(vehicle control) were applied to the mid-back of ten subjects for 24hours. The sites were then clinically assessed and, along with normalcontrol skin, surface cells were harvested with four applications ofindividual tapes and by shave biopsy. RNA was extracted from the tapesand biopsies and assayed semi-quantitatively for Il-1β, IL-8, GAPDH andβ-actin mRNA using fluorescent, quantitative RT-PCR. The results showedconsistent increases in IL-1β and IL-8 mRNA in inflamed skin relative tountreated skin. Furthermore, experiments disclosed in this Exampleillustrate the successful use of tape-harvested RNA to profile normaland experimentally inflamed skin using DNA microarrays. This profile ofSLS-irritated skin is an important step in the definition of RNAprofiles designed to differentiate irritant from allergic skinreactions.

Materials and Methods

Clinical protocols: The study protocols were reviewed and approved by anindependent IRB (BioMed IRB, San Diego) and all subjects signed informedconsent. Ten healthy women, ages 21–55 were enrolled in the study.Regions of unblemished, normal appearing skin on the mid back werechosen for the application of 2 occlusive patches in the form ofbandages approximately 4 cm×6.5 cm. The bandages were made using aclear, non-porous plastic hypoallergenic adhesive tape. In the center ofthis tape was a Webril (non-woven cotton) patch measuring approximately2 cm×4.5 cm. One Webril patch contained 0.6 ml of 1% aqueous sodiumlauryl sulfate and the other contained 0.6 ml of sterile water as thevehicle control. Patches were arranged such that the SLS patch wassuperior and directly adjacent to the water patch; the area of normalcontrol skin was inferior to and adjacent to the water patch. At 24hours post application, the SLS and water patches were removed and theskin allowed to air dry for 15 minutes before scoring. The sites werescored by a trained technician using the scale provided below. Patchedsites were large enough that two areas could be tape harvested withoutoverlap and room left for a shave biopsy (˜2×2 mm). Skin sites were tapestripped with 4 tapes each and then a shave biopsy taken under localanesthetic (lidocaine HCl 1% and epinephrine 1:100,000; AbbottLaboratories). The tapes were applied to the skin using 20 seconds offirm pressure with a circular motion The tape used for tape strippingwas a synthetic rubber-based adhesive on a polyurethane film (ProductNo. 90068, Adhesive Research, Glen Rock, Pa.). An area of uninvolvedskin was tape harvested and shave biopsied in an identical manner. Tapeswere stored in individual eppendorf tubes at −80 until extraction;biopsy samples were placed in buffer RLT and stored at −80 untilextraction. Skin responses to each patch application were examined andgraded under light supplied by a 100-watt incandescent blue bulb. Thefollowing grading scale was used: 0, no visible reaction; 1, slight,pink, patchy erythema; 2, mild confluent, pink erythema; 3, moderateerythema (definite redness) with edema; 4, strong erythema (very intenseredness) with edema. In a second study, 3 subjects had 3 patchescontaining 1% SLS and 3 water patches applied to the mid back for 24hours. Patches were removed, scored and tape stripped as above. In athird study, two individuals were tape stripped on uninvolved skin onthe upper back at three adjacent sites as above. RNA harvested in theselast two studies was used in the DNA microarray experiments describedbelow.

Materials and reagents: Adhesive tape was purchased from AdhesivesResearch, Inc. (Product No. 90068) (Glen Rock, Pa.) in bulk rolls. Theserolls were custom fabricated into small circular discs, 17 millimetersin diameter, by Diagnostic Laminations Engineering (Oceanside, Calif.).Total spleen RNA was purchased from Ambion. “RNeasy” RNA extraction kitand Sensiscript Reverse Transcriptase kit were purchased from Qiagen(Valencia, Calif.). PCR primers and probes (TaqMan™ Pre-Developed AssayReagents) and TaqMan Universal Master Mix, which included all buffersand enzymes necessary for the amplification and fluorescent detection ofspecific cDNAs, were purchased from Applied Biosystems (Foster City,Calif.). Total mRNA was amplified using the MessageAmp aRNA kitpurchased from Ambion Inc. (Austin, Tex.). Human Genome U133A DNA chipswere purchased from Affymetrix Inc. (Santa Clara, Calif.).

Isolation of RNA: The RNA within skin cells adherent to the 4 tapes usedto harvest a site was pooled by simultaneously extracting the tapes in avolume of buffer RLT (supplied with RNeasy kit). Extraction wasperformed using the manufacturer's directions and included a ProteinaseK digestion, sonication of tapes and “on-column” DNase I digestion. RNAwas eluted in 100 microliters of sterile, RNase free water. Extractionof biopsies was performed with the same kit according to themanufacturer's instructions.

Quantitative RT-PCR: 10 μl of RNA was reverse transcribed (RT) into cDNAwith the Sensiscript Reverse Transcriptase kit and random hexamers in afinal volume of 20 μl according to the manufacturer's directions. Thereaction was diluted 5-fold with sterile, nuclease-free water (Ambion)for use in the subsequent amplification/detection reaction. For eachspecific mRNA detection, 3 replicate RT⁺ reactions and one RT⁻ (noreverse transcriptase; negative control) reaction were performed. Twoamplification/detection reactions were done on each RT⁺ reaction toyield a total of 6 independent determinations of the threshold value(C_(t); discussed below). All RT⁻ reactions were amplified using 2replicates and were negative (data not shown).

Quantitation of RNA mass recovered with adhesive tape and biopsy: Theamount of RNA recovered by tape is too small (in most samples) to detectby UV. We have also found that contaminants in the adhesive co-purifywith the RNA and interfere with UV and fluorometric detection. Wetherefore estimated the RNA mass recovered from tapes by usingquantitative RT-PCR with reference to a standard curve (C_(t, actin) vs.log[RNA]; (AppliedBiosystems 2001)) created from commercially purchasedhuman spleen total RNA. Spleen RNA was treated with DNase I and purifiedwith the Qiagen RNeasy kit following the manufacturer's instructions.Purified standard RNA was quantified spectroscopically using O.D. 260.The standard curve was constructed using 4 concentrations of RNA from0.01 to 1 μgm/ml. Each RNA standard was reverse transcribed intriplicate and each RT reaction amplified once to yield 3 replicates perstandard concentration. Amplification and detection of unknowns wasaccomplished as described below using β-actin mRNA as the quantifiedmarker. Experimental samples were reverse transcribed in triplicate andeach RT reaction amplified in duplicate to yield a total of 6replicates. The average of these 6 replicates was used to calculate theconcentration of RNA in the unknown with reference to the standardcurve. Total RNA yields for all samples are reported in Table I. Theaccuracy of this method relies on the relative amount of β-actin mRNA tototal RNA in the epidermis being similar to that in human spleen. If therelative amount of β-actin mRNA to total RNA is different between thetwo tissues then our mass data will be similarly affected. Therefore, wedescribe all tape-harvested RNA mass calculations as estimates toreflect this uncertainty. RNA recovered from biopsies was quantifiedfluorometrically with the RiboGreen RNA Quantitation Reagent (MolecularProbes, Eugene, Oreg.).

Amplification and detection of specific mRNA: Specific mRNAs wereconverted to cDNA as described above. Specific cDNAs weresemi-quantified using gene specific primer/probes (5′-nuclease assay)and fluorescence detection. Amplification and detection assays wereperformed using TaqMan Pre-Developed Assay Reagents (PDAR; AppliedBiosystems) on an Applied Biosystems 7000 Sequence Detection System.β-actin, IL-1β and IL-8 mRNA assays were performed in the same tube(multiplex assay); these multiplex results were confirmed in repeatassays in single tube format (separate tube determination of β-actin andthe mRNA of interest; data not shown) using 6 replicates; GAPDH mRNAassays were done in singleplex format. Thermal cycling conditions were:prior to cycling, two minutes at 50° C., then ten minutes at 95° C.;then 40 cycles at 95° C. for 15 seconds and 60° C. for 60 seconds.Threshold detection was set at 0.2 for all assays.

Semi-quantitation of mRNA using the ΔΔC_(t) method: In this Example thecomparative or ΔΔC_(t) method of calculating relative gene expressionlevels between two samples, was used. In the ΔΔC_(t) method the levelsof IL-1β, IL-8 and GAPDH mRNAs are assayed semi-quantitatively bynormalization to β-actin mRNA to create a ratio of (mRNA_(x))/(β-actin)mRNA for each RNA sample. This ratio is then further normalized to acontrol sample (a process called “calibration”; (AppliedBiosystems2001)). When comparing the ratio of mRNA_(x) to β-actin mRNA between twodifferent samples, it is implicitly assumed that the level of β-actinmRNA to total RNA is constant between the two samples (i.e. it is anunchanging housekeeping gene). In the comparative method, the relativefold-increase of the mRNA of interest (“mRNA_(x)”) between 2 samples isgiven by the equation:

$\frac{\left( \frac{{mRNA}_{x}}{{mRNA}_{actin}} \right)_{Exp}}{\left( \frac{{mRNA}_{x}}{{mRNA}_{actin}} \right)_{Cal}} = 2^{{- {\Delta\Delta}}\; C_{t}}$where “Exp” indicates the experimental sample (in this case SLS or watersamples); “Cal” indicates the calibrator sample (uninvolved skin); andΔΔC_(t, x)=[ΔC_(t, Exp)−ΔC_(t, Cal)]_(x); where ΔC_(t, Exp) andΔC_(t, Cal) are calculated as (mean C_(t, RNAx))−(mean C_(t, actin)) forthe respective samples. The C_(t) values are the experimentallydetermined number of PCR cycles required to achieve a thresholdfluorescence (statistically significant increase in fluorescence overbackground) for mRNA_(x) and β-actin mRNA (Gibson, Heid et al. 1996;Heid, Stevens et al. 1996).

Calculation of fold-increase and data analysis: Key to the method usedto measure mRNA is the fact that the quantity of specific mRNA directlycorrelates with the number of cycles needed to reach thresholdfluorescence, thus the fewer the number of cycles (the lower the C_(t)),the more mRNA is initially present. As described above, experimentaldata is reported as the number of cycles (C_(t)) required to reach athreshold fluorescence. Each reported C_(t) is the mean of 6 replicatemeasurements. Calibrated fold-change calculations are made using theequations above. A mRNA_(x)/β-actin mRNA ratio was considered to have asignificant (with >95% confidence interval) fold-increase relative toits calibrator if the range of fold-change given by 2^(−(ΔΔCt±2 SEM))did not include the value of 1, which is the defined value of thecalibrator because the ΔΔC_(t) for the calibrator is equal to 0. Thesignificance of ΔC_(t) values (Tables I and IV) was determined byapplying a two-sided, paired t-test.

It is our experience that the practical limit of detection for real timePCR is 37 cycles. When the threshold number of PCR cycles (C_(t))extends beyond 37, these values become highly variable or fluorescencedoes not achieve a threshold value. To interpret combinations ofreplicate measurements of undetectable and >37 we applied the rule thatif 3 or more of the 6 replicates have any combination of C_(t) valuesequal to or greater than 37 (edge of detection) or are undetectable (noC_(t) value recorded), that mRNA is defined as undetectable and assigneda C_(t)=37. This assignment of a threshold equal to 37 typically occurswhen total RNA is low or when a message is not present (for instanceIL-1β and IL-8 mRNA in uninvolved skin). The assignment of C_(t)=37 tomRNAs that are undetectable is useful because it allows a calculation ofthe minimum fold-change of a mRNA_(x)/actin mRNA ratio between twosamples.

T7 linear RNA amplification: mRNA was amplified and biotin labeled usinga MessageAmp™ aRNA kit purchased from Ambion Inc. according to themanufacturer's instructions. Typical yields of aRNA obtained from tworounds of amplification ranged from 30–80 μg.

Hybridization of biotinylated mRNA targets to Affymetrix GeneChips,staining, data acquisition and data analysis: Hybridization and stainingwere performed according to the manufacturer's instructions. DataAquisition: Gene expression values from Affymetrix GeneChips are basedon the average difference (AD) between hybridization signals of perfectmatch (PM) and mismatch (MM) oligonucleotide probe sets for each gene asdescribed in the expression analysis technical manual from Affymetrix.The AD value of each probe set is calculated as AD=Σ(PM−MM)/# probepairs. In initial iterations, Affymetrix software removed probe pairsthat were out of a given range when calculating AD values for each probeset. In this process, the mean and standard deviation were calculatedfor intensity differences (PM−MM) across the entire probe set (excludingthe highest and lowest values), and values within a set number ofstandard deviations (3 as default) were not included in the calculation.The advantage was that this process minimized the variance introduced byexperimental or biological error by removing the outliners present ineach probe set. The disadvantage was that this process didn't alwaysremove the same probe pairs for the calculation of the AD values amongGeneChips. This led to the misinterpretation of the gene expressionprofiles obtained from GeneChip experiments. To alleviate this problem,a model-based method incorporated into a program called dChip wasdeveloped by Li & Wong (Li and Wong 2001). This method maintainsconstant probe pair set identities across all GeneChips while excludingoutliners due to cross hybridization, contamination duringhybridization, or manufacturing defects that affect probe setmeasurements. For all of the GeneChip experiments reported here, eachprobe pair set from the *.cel files were modeled by the dChip softwareprior to statistical analysis. The dChip-modeled expression measurementof each gene was normalized to the total signal of each chip. For anygiven measurement, a value greater than zero (indicating an expressionlevel) or a zero (indicating an expression level lower than background)was obtained. Only those genes exhibiting an expression level greaterthan zero in all experiments were used for statistical analysis. DataAnalysis: Many experimental designs and applications of gene expressionprofiling experiments are possible. However, no matter what the purposeof an experiment, each experiment must be replicated a sufficient numberof times for statistical analysis of the data. This is basically becauseeach gene expression profiling experiment results in the measurement ofthe expression levels of thousands of genes. In such a high dimensionexperiment, the chance for erroneous measurements for any individualgene expression level is high. Thus, in the absence of high replicationmany genes will show large changes in expression levels between twoexperimental conditions purely by chance alone, even when theexperimental conditions are the same. A simple t-test evaluates thedistance between the means of two groups normalized in terms of thewithin-group standard deviations. The result is that large differencesbetween genotypes for any given gene will be declared non-significant ifthe expression level of that gene is unreplicable within experimentaltreatments. Conversely, small differences in expression will bedetermined to be statistically significant for a given gene ifexpression levels for that gene are replicable within treatments. Inshort, the t-test statistic is constructed by scaling the difference ingene expression levels between genotypes relative to the observedvariances within genotypes. p-values based on the t-test statistic rangefrom 1.0 for gene expression levels with identical values associatedwith the null hypothesis to very small p-values for differential geneexpression levels that are highly significant. It must be noted,however, that the simple t-test does not perform well with a smallnumber of replicates. With a limited number of replicate measurements,often in the range of two to five for DNA microarray experiments, poorestimates of means, standard deviations, and p-values are obtained.

We have shown that the confidence in the interpretation of DNAmicroarray data with a low number of replicates can be improved by usinga Bayesian statistical approach that incorporates information of withintreatment measurements (Baldi and Long 2001; Long, Mangalam et al.2001). This results in a more consistent set of differentially expressedgenes identified with fewer replicates. The Bayesian approach is basedon the observation that genes of similar expression levels exhibitsimilar variance (Hatfield, Hung et al. 2003). Thus, more robustestimates of the variance of a gene can be derived by poolingneighboring genes with comparable expression levels. For the analysis ofthe data reported here we ranked the mean gene expression levels of eachreplicate experiment in ascending order, used a sliding window of 101genes, and assigned the average standard deviation of the 50 genesranked below and above each gene as the background standard deviationfor that gene. The variance of any gene within any given treatment wasestimated by the weighted average of the treatment-specific backgroundvariance and the treatment-specific empirical variance acrossexperimental replicates. In the Bayesian approach employed in thisstudy, the weight given to the within experiment gene variance estimateis a function of the number of experimental replicates. This leads tothe desirable property that the Bayesian approach employing such aregularized t-test converges on the same set of differentially expressedgenes as the simple t-test but with fewer replicates (Long, Mangalam etal. 2001).

While the Bayesian method provides more robust p-values, it must be keptin mind that these p-values represent the local confidence that can beplaced in an individual gene measurement. They say nothing about theglobal probability that an individual gene is differentially expressed.This can only be evaluated if an estimate of the global false positivelevel of each experiment can be determined. In other words, to interpretthe results of a high dimensional DNA array experiment it is necessaryto determine the global false positive and negative levels inherent inthe data set being analyzed. For this purpose we have implemented amixture-model based method described by Allison et al. (Allison, Gadburyet al. 2002) for the computation of the global false positive andnegative levels inherent in a DNA microarray experiment. The basic ideais to consider the p-values based on the regularized t-test describedabove as a new data set, and to build a probabilistic model for thesenew data. When control data sets are compared to one another (i.e. nodifferential gene expression) it is easy to see that the p-values willexhibit a uniform distribution between zero and one. In contrast, whendata sets from different genotypes or treatment conditions are comparedto one another (differential gene expression), a non-uniformdistribution will be observed in which p-values will tend to clustermore closely to zero than one; that is, there will be a subset ofdifferentially expressed genes with “significant” p-values. Thecomputational method of Allison (Allison, Gadbury et al. 2002) is usedto model this mixture of uniform and non-uniform distributions todetermine the probability, PPDE(p) ranging from 0 to 1, that any gene atany given p-value is differentially expressed; that is, that it is amember of the uniform (not differentially expressed) or the non-uniform(differentially expressed) distribution. With this method, we canestimate the rates of false positives and false negatives as well astrue positives and true negatives at any given p-value threshold,PPDE(<p). In other words, we can obtain a posterior probability ofdifferential expression PPDE(p) value for each gene measurement and aPPDE(<p) value at any given p-value threshold based on theexperiment-wide global false positive level and the p-value exhibited bythat gene. It should also be emphasized that this information allows usto infer the genome-wide number of genes that are differentiallyexpressed; that is, the fraction of genes in the non-uniformdistribution (differentially expressed) and the fraction of genes in theuniform distribution (not differentially expressed).

Commonly used software packages for microarray data analysis do notpossess algorithms for implementing Bayesian statistical methods.However, our statistical program, Cyber-T (www.igb.uci.edu) doesaccommodate this approach as well as the PPDE analysis described above.For the experiments reported here, we used these statistical toolsincorporated into Cyber-T.

Results

Total RNA Yields

RNA was recovered from 27 of 30 skin sites using 4 tapes as describedabove. The amount of total RNA recovered was variable from site to siteand subject to subject (data not shown). The average mass of RNArecovered from uninvolved skin sites was 0.92 nanograms (±0.35) with arange of 0 (2 samples) to 3.2 ng. The average mass of RNA recovered fromwater-occluded skin was 0.69 ng (±0.27) with a range of 0 (1 sample) to2.7 ng. SLS inflamed skin produced the greatest average yield of RNAwith an average of 185 ng (±76) and a range of 0.067 to 747 ng.

Relative Levels of Housekeeping Genes in Tape Strip Samples and Biopsies

Markers of the inflammatory process IL-1β and IL-8 mRNAs were chosen.Differential recovery of total RNA mass in a sample was accounted for bynormalizing these mRNAs to an internal control, the β-actin transcript.We then calibrated the mRNA_(x)/actin ratio in SLS and water samples tothat ratio in untreated skin samples. In this study, IL-1β and IL-8 mRNAare predicted to increase relative to β-actin in response to SLStreatment, while the level of housekeeping mRNAs, such as β-actin andGAPDH are predicted to remain constant. We have tested this assumptionby measuring the relative ratio of two housekeeping mRNAs, GAPDH andβ-actin, to determine if their ratio is indeed a constant in differentskin samples.

The measure of the relative abundance of two mRNAs in a sample is givenby the ΔC_(t) value, which is the difference between the experimentallydetermined threshold values. To investigate the possibility thatΔC_(t, GAPDH) (C_(t, GAPDH)−C_(t, actin)) may have unique values fordifferently treated skin samples, we determined these values for allsamples. Data in Table I show that biopsy-harvested RNA samples fromwater and SLS-treated sites have significantly different ΔC_(t, GAPDH)values (2-sided, paired t-test; p<0.005) than biopsy-harvesteduninvolved skin.

The data in Table I also demonstrate that tape-harvested RNA samplesfrom SLS-treated sites have significantly different ΔC_(t, GAPDH) valuesthan tape-harvested samples from water treated sites (p<0.005). Acomparison of tape-harvested samples from SLS treated and uninvolvedskin sites nears significance (p=0.087). Tape-harvested RNA samples fromwater-occluded sites do not have significantly different ΔC_(t, GAPDH)values than uninvolved skin (p=0.61).

Table I further compares the ΔC_(t, GAPDH) values between tape andbiopsy harvested samples of identically treated skin sites. The datashow that each sampling method produces an RNA sample with a differentΔC_(t, GAPDH), and that this difference is highly significant (p<0.005)for SLS-treated skin samples and uninvolved skin samples (p=0.014).Because biopsy and tape-harvested RNA samples have differingΔC_(t, GAPDH) values (and therefore different GAPDH/β-actin mRNAratios), we hypothesize that the sampling methods are recoveringdifferent cell populations.

The data in Table I can be used to calculate the fold-change inGAPDH/actin mRNA ratios relative to uninvolved skin (see Materials andMethods). The results of such calculations, shown in Table II, revealthat while there is some variation in the GAPDH/actin mRNA ratios fordifferent samples, the average variation amongst subjects for aparticular treatment is less than 2-fold. While some individual changesare greater than 2-fold, these differences are insufficient to explainthe much larger fold-changes we observe for IL-1β and IL-8/actin mRNAratios. Thus, while there are statistically significant changes inGAPDH/actin mRNA ratios due to water and SLS treatment of the skin,these differences do not explain the changes in IL-1β and IL-8/actinratios discussed later.

IL-1β/β-actin mRNA Ratios in SLS-irritated and Control Skin

Table III reveals the fold change of IL-1β/β-actin mRNA inwater-occluded and SLS-occluded skin relative to (calibrated to)uninvolved skin, in tape and biopsy harvested RNA samples. In 9 of 10biopsy samples of SLS-occluded skin, the IL-1β/β-actin mRNA ratio wassignificantly elevated. In 7 of 10 uninvolved skin biopsy samples, IL-1βmRNA was undetectable, while in the remaining 3 samples it was presentat very low levels (within 3 C_(t) units of our detection limit; datanot shown). In biopsy samples of water-occluded skin, IL-1β mRNA was notdetectable in 2 samples and was significantly elevated in 3 samples(Table III). Thus SLS-occlusion produced the most consistent elevationof the IL-1β/actin mRNA ratio but water-occlusion could effect a similaralbeit smaller response. When the effect of water-occlusion is takeninto effect by calibration of the SLS sample to the water-treated site,we find that 7 SLS-treated samples have significant increases in theIL-1β/actin mRNA ratio (data from Table III; calculation not shown).

Data in Table III reveal the fold change of IL-1β/β-actin mRNA intape-harvested samples of water-occluded and SLS-occluded skin relativeto uninvolved skin. Tape-harvested samples of SLS-occluded sites showedsignificant increases in 5 of 10 samples. These 5 samples with IL-1βincreases are in qualitative agreement with the biopsy data. Analysis ofthe remaining 5 samples was indeterminate due to low RNA recovery.Similar to biopsy samples, tape samples of uninvolved skin did not havedetectable amounts of IL-1β with one exception. Tape-harvested samplesof water-occluded skin also showed undetectable amounts of IL-1β mRNA in9 of 10 samples, while one sample with detectable IL-1β displayed asignificant increase in IL-1β/actin mRNA relative to uninvolved skin.

IL-8/β-actin mRNA Ratios in SLS-irritated and Control Skin

Table III reveals the fold increase of the IL-8/β-actin mRNA ratio inwater-occluded and SLS-occluded skin compared to uninvolved skin. Thedata demonstrate that 8 of 10 biopsy samples of SLS-occluded skinrevealed significant increases in IL-8/actin mRNA ratios. Biopsy samplesfrom 9 of 10 untreated skin sites had undetectable IL-8 mRNA levels. Theone uninvolved skin biopsy sample with detectable IL-8 mRNA was close tothe level of detection. Thus IL-8 mRNA was generally not detectable in abiopsy of uninvolved skin. Similarly 5 of 10 samples from biopsies ofwater-occluded sites also had undetectable IL-8 mRNA. In the remaining 5water-treated samples, 2 had significantly increased IL-8/actin mRNAratios although these increases were small in comparison to therespective SLS-occluded sites. Thus water-occlusion did not appreciablystimulate IL-8 mRNA appearance in the epidermis of most subjects.

Table III further reveals that 8 of 10 tape-harvested samples fromSLS-occluded sites displayed significantly increased IL-8/β-actin mRNAratios. Of the 2 samples without significant increases, one did not havedetectable IL-8 mRNA (the sample was low in RNA and the result isinconclusive) while the second sample (Subject 4) likely had increasedIL-8 message (see Discussion) but this could not be confirmed becausethe control tape sample failed to recover RNA. Data in Table III showthat tape-harvested samples of water-treated sites reveal significantincreases in IL-8/actin mRNA ratios in 3 subjects. In the remainingsubjects, IL-8 could not be detected at significantly elevated levels.Thus, the tape data is in good qualitative agreement with the biopsydata with a majority of inflamed sites revealing increases in IL-8 mRNA.

Table IV reveals ΔC_(t, IL-8) values for all samples. This data supportsand extends the previous observation that biopsy and tape harvestedsamples of equivalently treated sites may produce significantlydifferent ΔC_(t) values. Table IV reveals that the average ΔC_(t, IL-8)value from tape-harvested, SLS-treated sites was −0.89 while the averagevalue from biopsy harvested SLS sites was 4.13. A paired t-test betweenthe individual ΔC_(t) values has a p<0.005. A similar comparison ofΔC_(t, IL-8) values between biopsy and tape harvested RNA samples fromthe water-occluded sites also shows a highly significant difference(p<0.005). These observations extend to uninvolved skin as well. TableIV shows that 4 samples of tape-harvested uninvolved skin hadΔC_(t, IL-8) values with a range of 1.51 to 4.15 while the analogousbiopsy samples had a range of 8.22 to >9.7, a clear difference. Theability to consistently detect higher amounts of IL-8 mRNA in normal,water and SLS treated skin samples recovered by tape reinforces thehypothesis that tape harvesting preferentially recovers a subset ofcells (probably close to the surface) poorly represented in biopsies.

DNA Microarray Analysis of RNA Extracted from Uninvolved Skin Using Tape

The success in the above analysis of tape-harvested RNA from differentskin sites suggested that this RNA might be amenable to amplificationand hybridization to DNA microarrays. In order to assess thereproducibility and consistency of tape-harvested RNA samples for geneexpression profiling experiments, three samples were collected from theupper back of each of two healthy individuals, one male (sample C1, C2,and C3) and one female (sample A5, A6, and A9). Approximately onenanogram of total RNA was isolated and the mRNA was amplified and biotinlabeled using a MessageAmp™ aRNA kit as described in Materials andMethods. The resulting biotin-labeled aRNA from each sample was used forhybridization to an Affymetrix HG-U133A GeneChip.

The results in TABLE V show the differences observed when a matrix ofpair-wise gene expression comparisons between two GeneChips wasperformed using Affymetrix Microarray Suite software. These data show anaverage of only 12% variance among gene measurements, regardless ofwhether data from different sites on the same individual or sites fromdifferent individuals are compared. Furthermore, comparing the data inquadrant three of TABLE V (A vs. C) to the data in quadrants one (A vs.A) and four (C vs. C) shows that about 15% of this variance is due toeither gender difference (A vs. C) or inter-subject variation (A vs. Aor C vs. C). Thus, amazingly little variance is contributed by samplesobtained from different sites or from different individuals.

To compare these data in a more quantitative manner, the threeAffymetrix GeneChips each hybridized with targets from RNA samplesobtained from individual A were compared to three GeneChips hybridizedwith targets from the three RNA samples obtained from individual C.These data were analyzed with a regularized t-test implemented in theCyber-T statistical program. This three-by-three comparison revealed21,790 probe sets that exhibited gene expression levels above backgroundfor all three sites from each subject. Of these genes 1,117 (5%) weredifferentially expressed with p-value less than 0.0035, which based onthe global false positive and negative levels of this data setcorresponds to a posterior probability of differential expression (PPDE)value of 0.95. Thus, 56 of the 1,117 differentially expressed genes thatexceed this p-value threshold are expected to be false positives. Thesource of these inter-subject gene expression differences remains to bedetermined, however at least one of these differences is gender based.For example, the gene with the smallest p-value and the highest PPDEvalue is the Y-linked ribosomal protein S4 (PRS4Y). It is likely thatdifferences that are not gender based are a reflection of normalvariation of gene expression between individuals. These data areavailable at www.igb.uci.edu.

DNA Microarray Analysis of Normal Versus Water-occluded and SLS-occludedSkin.

In a separate experiment, a total of nine RNA samples ranging from 1–10nanograns were isolated by tape harvesting from three untreated, threewater-occluded, and three SLS-occluded sites of each of threeindividuals. mRNA from each of the nine samples was amplified, biotinlabeled and used for hybridization to each of nine Affymetrix HG-U133AGeneChips as shown in FIG. 1.

Untreated vs. SLS treated samples. A comparison of gene expressionlevels between three untreated (A1, B1, C1) samples and three SLStreated (A2, B2, C2) samples revealed 21,031 genes that exhibitedexpression levels above background for all samples. To assess theconfidence in global changes in gene expression, the p-values for allgene measurements were distributed into 100 bins ranging from 0 to 1.0and plotted against the number of genes in each bin (FIG. 2A). Theβ-mixture modeling methods implemented in Cyber-T were used to modelthese p-value distributions of the uniform (not differentiallyexpressed) and non-uniform (differentially expressed) data sets todetermine the posterior probability of differential expression, PPDE, ofeach gene based on global false positive and negative gene measurementlevels as described by Hung et al. (Hung, Baldi et al. 2002) and Baldiand Hatfield (Baldi and Hatfield 2002). When untreated vs. SLS occluded(FIG. 2A) data are compared, the p-values for the differentiallyexpressed genes are low and cluster toward 0. This is consistent withhighly statistically significant differences among measurement levels ofsome genes. In fact 1,771 genes that are differentially expressed with athreshold of p=0.003, which corresponds to a PPDE(p) value equal to orgreater than 0.99. These data are available in Table VII provided on thecompact disk filed herewith as file 6392999.xls.

SLS vs. water treated samples. A comparison of gene expression levelsbetween three SLS treated (A2, B2, C2) samples and three water treated(A3, B3, C3) samples revealed 21,307 genes that exhibited expressionlevels above background for all samples. The p-values for all of thesegene measurements were also distributed into 100 bins ranging from 0 to1.0 and plotted against the number of genes in each bin (FIG. 2B). It isevident from an examination of the p-value distribution that, similar tothe comparison with untreated cells, about twenty percent of the genesare differentially expressed. Based on a threshold of p=0.003, 1,364genes are differentially expressed with a PPDE(p) value of 0.99. Ofthese, 1,063 genes are also differentially expressed with a p-value of0.003 and a PPDE value of 0.99 when SLS and untreated samples arecompared. These data are available at www.igb.uci.edu.

Untreated vs. water treated samples. A comparison of gene expressionlevels between three untreated (A1, B1, C1) samples and three watertreated (A3, B3, C3) samples revealed 21,164 genes that exhibitedexpression levels above background for all samples. The p-values for allthese gene measurements were again distributed into 100 bins rangingfrom 0 to 1.0 and plotted against the number of genes in each bin (FIG.2C). The fact that these p-values are uniformly distributed demonstratesthat, at the levels of variance inherent in these experiments, there areno statistically significant differences between the gene expressionlevels of these two data sets. Nevertheless, based on a review of thegenes assigned the lowest p-values, many of which are associated withinflammation, we believe that the water treatment does lead to somechanges in gene expression compared to untreated control skin. Thesedata are found in Tables VII (provided in the attached Appendix) andVIII (See attached Compact Disk).

For purposes of discussion, only the 100 genes differentially expressedwith p-values less than 1.4×10⁻¹⁰ and PPDE(p) values greater than 0.99are discussed here and in TABLE VI. An examination of these top 100genes most significantly altered when the SLS treated skin samples werecompared to untreated skin samples revealed that, as expected, most ofthese genes carry out functions related to tissue inflammation andinjury (TABLE VI). These differentially expressed genes are proteinases,protease inhibitors, cytokines, chemokines, complement components, HLAfactors, or receptors involved in immune regulation. These associationswith inflammation and injury responses for many of these mostlyup-regulated genes are documented in the literature (TABLE VI). Theseresults demonstrate that the tape stripping method described hereharvests RNA suitable for complete gene expression profiles of the skinthat accurately reflect its pathological state.

Discussion

Recent advances in molecular medicine have made the possibility ofmolecular diagnosis a reality (Aitman 2001; Bertucci, Houlgatte et al.2001; Galiegue and Casellas 2002; Lacroix, Zammatteo et al. 2002;Whipple and Kuo 2002; Satagopan and Panageas 2003). Through the use ofmicroarrays and RNA profiling it is becoming increasingly clear thatsimple and complex cell populations can be monitored or “profiled” withthe intent of understanding the physiologic state of those cells ortissues. This information is expected to lead to more accurate andpossibly predictive diagnoses. This Example illustrates that the use of4 small tape strips is an effective and non-invasive approach tocapturing messenger RNA from the surface of skin and that this techniquepermits a direct quantitative and qualitative assessment of pathologicand physiologic biomarkers as a function of normal physiology.

The levels of IL-1β and IL-8 mRNA have been assayed semi-quantitativelyrelative to β-actin in normal, water and SLS-occluded skin sites andshown that RNA from tape and biopsy samples produce qualitativelysimilar results. In order to account for the possibility that changes inβ-actin mRNA were responsible for observed changes in theinterleukin/β-actin mRNA ratios, (Suzuki, Higgins et al. 2000; Bustin2002; Tricarico, Pinzani et al. 2002) the levels of two housekeepinggenes relative to each other were quantified. The resulting data (TablesI and II) showed that while the GAPDH/β-actin mRNA ratio is different indifferently treated skin samples, the magnitude of this difference isnot capable of explaining the observed changes in Il-1β and IL-8 mRNAlevels. This fact is most clearly demonstrated by tape-harvest andbiopsy data in which IL-1β and IL-8 mRNA are virtually undetectable incontrol samples but easily detected in SLS-treated samples, anobservation that cannot be explained by minor changes in β-actin mRNAlevels. In addition, IL-1β and IL-8 mRNAs and proteins have been wellcharacterized in inflammation and are known to become elevated inresponse to SLS and other treatments (Paludan and Thestrup-Pedersen1992; Grangsjo, Leijon-Kuligowski et al. 1996; Corsini and Galli 1998;Tomic-Canic, Komine et al. 1998; Freedberg, Tomic-Canic et al. 2001;Perkins, Osterhues et al. 2001; Cumberbatch, Dearman et al. 2002;Coquette, Bema et al. 2003). Thus data provided herein, from both tapeand biopsy, are consistent with published observations.

Biopsy and tape harvesting are not equivalent sampling methods andtherefore should not be expected to yield identical results. Tapeharvest is restricted to the skin surface and therefore maypreferentially recover vellus hair follicles and cells lining sebaceous,eccrine and sweat ducts as well as corneocytes (not predicted to containRNA). Our method of using a single application of 4 individual tapesdoes not result in glistening of uninvolved skin and thus does not barethe viable epidermis. In contrast, a shave biopsy is expected to includenot only cells of the epidermis (primarily keratinocytes and melanocytesand immune cells) but fibroblasts from the upper dermis. The potentialenrichment of surface epidermis conveyed by our circular tape comparedto a shave biopsy can be appreciated by considering that the surfacearea of a tape is 284 mm², while the surface area of a 2×2 mm shavebiopsy is 4 mm². Thus we propose that tape-harvested cells represent anenrichment of a sub-population of cells found in a shave biopsy.

The data presented in Tables I and IV support the hypothesis that tapeand biopsy-harvested RNA are derived from different cell populations.Table I shows highly significant p values when comparing ΔC_(t, GAPDH)values between tape and biopsy samples of SLS and uninvolved skinsamples. Table IV demonstrates that ΔC_(t, IL-8) is highly significantlydifferent (p<0.005) between tape and biopsy samples derived from normal,water or SLS-treated skin samples. In SLS-treated skin samples, thedifference in average ΔC_(t, IL-8) values implies that IL-8/β-actin mRNAratio is 2^(−(−0.89-4.13)) or 32-fold greater in tape versusbiopsy-harvested RNA samples. In water-treated samples, the IL-8/β-actinmRNA ratio is on average 2^(−(1.54-9.22)) or at least 200-fold greaterin tape-harvest RNA samples (data from Table IV). Similar, supportivedata was also observed for ΔC_(t, IL-1β). This data implies that somedifferentially expressed biomarkers may be best detected in tape ratherthan biopsy-harvested epidermal samples.

Identification of biomarkers diagnostic of clinical irritation has beena long sought goal (Muller-Decker, Furstenberger et al. 1994; Boelsma,Gibbs et al. 1998; Muller-Decker, Heinzelmann et al. 1998; van Ruissen,Le et al. 1998; Komine, Rao et al. 2001; Perkins, Osterhues et al. 2001;Boxman, Hensbergen et al. 2002; Perkins, Cardin et al. 2002; Coquette,Bema et al. 2003). Changes in IL-1β and IL-8 mRNA are used herein asindicators of irritation and shown that most but not all irritated sitesdisplay increased levels of these normalized mRNA markers (Table III).The data also shows that tape-harvested and biopsy recovered RNA arequalitatively equal in their ability to reveal an irritant skinreaction. With respect to biopsy samples, it is clear that neithermarker is 100% efficient at diagnosing irritation, a result observed forevery biomarker proposed to be diagnostic of erythema and inflammation(Grangsjo, Leijon-Kuligowski et al. 1996; Muller-Decker, Heinzelmann etal. 1998; Chung, Marshall et al. 2001; Perkins, Osterhues et al. 2001;Boxman, Hensbergen et al. 2002). The current limitation of the tapeharvest assay is the inefficiency in detecting certain markers insamples with limiting amounts of RNA, a subject discussed below.However, in comparison with the Sebutape assay (immunoassay of IL-8protein; (Perkins, Osterhues et al. 2001) for irritation, which has asensitivity (Hoffrage, Lindsey et al. 2000) of approximately 30%, mRNAbiomarkers seem to possess superior potential. The observation thatwater occlusion produced increases of biomarker ratios in some subjectshas been reported by others (Grangsjo, Leijon-Kuligowski et al. 1996;Howie, Aldridge et al. 1996; Perkins, Osterhues et al. 2001).

Results presented herein show that the tape stripping method harvestsRNA suitable for DNA microarray experiments, and that these geneexpression profiles reflect the pathological state of human skin, itshould be possible to identify a subset of genes whose differentialexpression patterns can be correlated with different pathological stateswith a high degree of statistical accuracy. The fact that 1,700differentially expressed genes have been identified with highstatistical confidence sets the stage for the creation of small customDNA arrays designed to identify patterns of gene expression diagnosticof irritant skin reactions, possibly diagnostic of different irritantsand predictive of irritant reactions. The next step along this path isto identify the analogous set of genes expressed during an allergic skinresponse, identify genes unique to the irritant or allergic response andcombine them into one DNA array, which could be used to determine if amild reaction to a substance is irritant or allergic in nature. Such anarray could also be used to test a variety of irritants and allergensfor unique profiles.

Analysis of the top 100 genes differentially expressed in ourSLS-treated samples shows that well over half of these genes have beenimplicated in injury and inflammation (TABLE VI), with most of thesegenes being up regulated. Interestingly, many of the down-regulatedgenes are hair keratins and keratin associated proteins selectivelyexpressed in the hair during the anagen phase of the hair cycle. Either,the occlusive SLS treatment removes hair prior to the tape stripping orthe treatment blocks anagen in the hair follicles.

It is shown in this example that RNA can be non-invasively andproductively recovered from the surface of the skin using 4 small tapestrips. The number of tape strips can be reduced, for example to twotape strippings, in conditions where the surface of the skin has beendisrupted, such as SLS occlusion for 24 hours or in hyperproliferativeskin conditions such as psoriasis (See Example 2). Furthermore, thelimitation of capturing small amounts of RNA from some skin sites can beeffectively overcome by obtaining replicate control samples and by theappropriate choice of mRNA biomarker (discussed below). Also presentedherein, is data that the ΔC_(t) value, which is normally used tocalculate a ΔΔC_(t) value (and thus a calibrated fold-change), is itselfpotentially useful for characterizing the physiologic state of theepidermis without reference to a calibration site.

The potential utility of ΔC_(t) values is illustrated by theΔC_(t, IL-8) for subject 4's SLS-treated skin (tape-harvested sample;Table IV). That ΔC_(t) is −1.28, however it cannot be used to calculatea ΔΔC_(t) value (and therefore a fold-change) because insufficient RNAwas recovered from the normal and water-occluded control sites. However,comparison of this ΔC_(t) value to the remaining subjects' average SLSΔC_(t, IL-8) value of −0.89 and average values from tape-harvestedwater-occluded and uninvolved skin sites (>2.49 for normal or 1.54 fortape) is highly suggestive that the ΔC_(t) value of −1.28 is in factindicative of irritated skin. For example, the value of −1.28 impliesthat, compared to the average value for the 10 subjects, subject 4'sSLS-site IL-8/β-actin mRNA ratio is at least 2^(−(−1.28-2.49)) or13.6-fold higher than the average value for uninvolved skin. A similarcalculation using the average ΔC_(t, IL-8) for water-occluded samples asthe calibrator suggests that the IL-8/actin mRNA ratio is2^(−(−1.28-1.54)) or 7.1-fold elevated. These data lead to thehypothesis that establishment of a database of ΔC_(t) values fordifferent mRNA biomarkers might be useful to identify a physiologic skinstate without reference to an intrasubject control site. This utility ofΔC_(t) values is predicated upon the consistency of the PCR reactionconditions and the use of identical probes between samples. Given theseprerequisites, our data support the potential for ΔC_(t) values beingdiagnostic indicators.

In this study the quantity of RNA recovered from different individualsand skin sites was variable, with significantly more RNA being recoveredfrom SLS-treated sites than uninvolved skin sites. The large amount ofRNA recovered from the SLS-irritated sites is consistent with the knowneffects of SLS, which effects invasion of inflammatory cells and createsa weakened barrier facilitating the removal of the inflamed epidermis.It has been found that RNA recovery is also a function of anatomicalsite and similar sites vary between individuals with respect to RNAyield.

While the variability of total RNA recovered does not affect the resultsof relative gene quantitation, the recovery of very small amounts of RNAdid affect our ability to fully analyze some samples. In this respect,the choice of biomarkers may be as important as the amount of RNArecovered from a site. For instance, Table I shows that mosttape-harvested samples could be assayed for β-actin and GAPDH mRNAs andthus calibrated GAPDH/actin ratios could be calculated. However, TableIII reveals that some of these same samples do not have calibratedIL-1β/actin or IL-8/actin mRNA ratios, with the IL-1β assay being themost affected. The reason for this difference between biomarker assayslies in the relative abundance of the specific mRNA. Because GAPDH mRNAis approximately equal in abundance to β-actin mRNA, all samples withdetectable actin mRNA were successfully assayed for GAPDH. Likewise, ahigh success rate was achieved at calibrating IL-8/actin mRNA ratios inwater and SLS treated tape-harvested skin samples because IL-8 messageis relatively abundant in these samples. Thus the biomarker mRNA that isthe most abundant will make the most efficient use of RNA mass.Therefore, candidate biomarker mRNAs should be chosen for bestsensitivity, positive predictive value and high relative abundance whenRT-PCR is to be used for detection and tape harvesting is to be thesampling method.

The present Example demonstrates the utility of tape-harvested RNA forsemi-quantitative RT-PCR and microarray applications for severalreasons. Both methods have particular advantages and are appropriate indifferent circumstances. The use of microarrays is an invaluable toolfor the discovery of diagnostic and prognostic biomarker candidates andmay be essential for subcategorizing disease states, which may demandsimultaneous assay of hundreds of biomarkers. However, the use ofmicroarrays is expensive and technically laborious. Quantitative RT-PCRis less expensive and less technically demanding and is appropriate forstudies where a limited number of known markers are being studied.

In summary, the data of this Example show that the tape stripping methodcollects skin samples from normal and inflamed skin that are suitablefor RNA isolation and gene expression profiling experiments. This methodcan be used to profile expression of a large number of genes indifferent skin conditions to design custom arrays that allow moleculardiagnoses of skin disorders.

TABLE I ΔC_(t,GAPDH) values in tape and biopsy-harvested RNA samplesfrom treated and untreated skin. ΔC_(t,GAPDH) ^(a) Tape Biopsy SubjectNormal Water SLS Normal Water SLS 1 3.27 ± 0.36 2.04 ± 0.33 2.78 ± 0.44  0.1 ± 0.0.06 0.94 ± 0.09 1.61 ± 0.11 2 1.23 ± 0.17 1.41 ± 0.12 2.56 ±0.11 0.99 ± 0.07 1.79 ± 0.12 1.95 ± 0.08 3 0.86 ± 0.15 1.54 ± 0.18 3.02± 0.08 0.73 ± 0.10 1.41 ± 0.11 1.44 ± 0.11 4 — — 2.28 ± 0.10 0.46 ± 0.160.95 ± 0.11 1.68 ± 0.09 5 — 2.04 ± 0.23 3.15 ± 0.15 0.83 ± 0.10 1.69 ±0.10 1.78 ± 0.10 6 2.97 ± 0.15 2.71 ± 0.23 2.87 ± 0.10 0.24 ± 0.07 1.48± 0.06 2.33 ± 0.07 7 0.91 ± 0.13 1.47 ± 0.10 2.68 ± 0.09 0.31 ± 0.10   1± 0.07 1.73 ± 0.09 8 1.83 ± 0.13 1.04 ± 0.15   3 ± 0.11 0.04 ± 0.04 1.38± 0.12 1.54 ± 0.10 9  0.7 ± 0.19 2.16 ± 0.34 2.85 ± 0.15 0.35 ± 0.08 1.6 ± 0.10 3.25 ± 0.08 10  3.47 ± 0.15 1.01 ± 0.12 2.46 ± 0.08 0.87 ±0.09 1.77 ± 0.12 1.05 ± 0.07 Average 1.91 1.67 2.78 0.49 1.4 1.84 (SD)(1.35) (0.35) (0.04) (0.34) (0.33) (0.6) p value^(b) — 0.61 0.087 —<0.005 <0.005 (vs. normal) p value^(c) 0.61 — <0.005 <0.005 — 0.06 (vs.water) p value^(d) 0.014 0.28 <0.005 — — — (biopsy vs. tape)ΔC_(t,GAPDH) is defined as [C_(t,GAPDH) − C_(t,actin)]; see Materialsand Methods; mean ± SD; tape-harvested samples from uninvolved skin ofsubjects 4 and 5 and water-occluded skin (subject 4) did not havesufficient RNA for accurate β-actin assay. The p value (2-sided, pairedt-test) for water vs. normal and SLS vs. normal for biopsy andtape-harvested samples. The p value for water vs. normal and water vs.SLS for biopsy and tape-harvested samples. The p value fortape-harvested versus biopsy samples for normal, water and SLS-occludedskin.

TABLE II GAPDH/actin mRNA ratios in EGIR and biopsy samples of normal,water and SLS-occluded skin. Relative GAPDH/β-actin mRNA change^(a) EGIRBiopsy Subject SLS Water SLS Water  1 [1] 1.41 2.35 0.35 0.56  2 [3] 0.40.88 0.51 0.58  3 [3] 0.22 0.62 0.61 0.62  4 [2] — — 0.43 0.72  5 [3] —— 0.52 0.55  6 [4] 1.07 1.19 0.24 0.42  7 [3] 0.29 0.68 0.38 0.62  8 [3]0.44 1.72 0.35 0.4  9 [3] 0.22 0.36 0.13 0.42 10 [4] 2.02 5.48 0.88 0.54Average 0.55 1.18 0.44 0.54 ^(a)Fold-increase of GAPDH/β-actin mRNA inthe indicated sample relative to uninvolved skin; individualfold-changes calculated from data in Table I and average changescalculated from average values in Table I as described in Materials andMethods.

TABLE III Summary of fold-changes in IL-1β/β-actin and IL-8/β-actin mRNAratios relative to uninvolved skin. IL-1β/β-actin IL-8/β-actin Tape^(b)Biopsy^(b) Tape^(b) Biopsy^(b) Subject^(a) Water SLS Water SLS Water SLSWater SLS 1 [1] ND ND   3.7**    1 ND ND ND  >1.1 2 [3] 4.5** >7.3**^(†) >1.4 >28**^(†)    0.85    6**^(†) ND    84**^(†) 3 [3] ND  18**^(†) ND >15**^(†)    1.8    24**^(†) ND >171**^(†) 4 [2] — NC >2.7 >3.3** — NC ND  >12**^(†) 5 [3] — NC ND  >8**^(†)  >1.1  >9.3**^(†) >1.5 >101**^(†) 6 [4] ND  >0.44 >6.7** >34**^(†)  >6.3**  >6.8** >10**>778**^(†) 7 [3] ND  >8.1**^(†) >3.1 >11**^(†) >24** >235**^(†)  >1.4 >42**^(†) 8 [3] ND  >1.9**   2.3   56**^(†)  >1.4  >41**^(†)  >1.9>594**^(†) 9 [3] ND  >5** >2 >53**^(†)   22**    18**  >5.2** >368**^(†)10 [4]  ND  >1.4   2.2**    2.2**    1.3  >3** ND  >1.1 ^(b)Subjectidentification followed by clinical score in brackets, score definitionsare discussed in Materials and Methods. ^(c)Fold-change is calculatedfrom mean Ct values (IL-1β data not shown and IL-8 data in Table IV) asdescribed in Materials and Methods. The following abbreviations areused; “—” indicates insufficient RNA recovered to accurately assayβ-actin mRNA; “ND” indicates IL-1β or IL-8 mRNA was not detected in theindicated sample (water or SLS); NC indicates that the mRNA could bedetected in the indicated sample but a calculation of fold-change couldnot be made due to low RNA recovery in the uninvolved skin sample; a“**” signifies that the fold-change is significant at greater than 95%confidence; a † designates that the fold-change is also significant whencalculated relative to the water sample (data not shown); a > symbolindicates that IL-1β or IL-8 mRNA could not be detected in the controlsample, thus a minimum estimate of fold-change was calculated asdescribed in Materials and Methods.

TABLE IV ΔC_(t) values for IL-8 mRNA in normal, water-occluded andSLS-occluded skin. ΔC_(t,IL-8) ^(b) Normal Water SLS Subject^(a) TapeBiopsy Tape Biopsy Tape Biopsy 1 [1]   1.51 ± 0.37  >9.26 ± 0.06 >1.28 ±0.11   >9.68 ± 0.13   >1.51 ± 0.17 9.15 ± 0.15 2 [3]   2.23 ± 0.17   8.22 ± 0.22 2.46 ± 0.19 >11.30 ± 0.07    −0.36 ± 0.05 1.82 ± 0.05 3[3]   2.08 ± 0.16  >9.71 ± 0.06 1.26 ± 0.28 >10.76 ± 0.11    −2.59 ±0.07 2.29 ± 0.13 4 [2] — >10.16 ± 0.10 — >10.53 ± 0.10    −1.28 ± 0.116.56 ± 0.10 5 [3] —  >9.72 ± 0.05 0.03 ± 0.27 9.09 ± 0.31 −3.05 ± 0.153.06 ± 0.11 6 [4]  >2.7 ± 0.08 >10.36 ± 0.09 0.05 ± 0.13 7.00 ± 0.27−0.05 ± 0.11 0.76 ± 0.13 7 [3] >7.20 ± 0.13 >10.52 ± 0.06 2.62 ± 0.1410.06 ± 0.28  −0.68 ± 0.08 5.11 ± 0.06 8 [3] >4.91 ± 0.18 >10.89 ± 0.084.46 ± 0.32 10.00 ± 0.4  −0.44 ± 0.06 1.67 ± 0.08 9 [3]   4.15 ± 0.28 >9.58 ± 0.09 −0.30 ± 0.18   9.94 ± 0.28   0.02 ± 0.17 1.06 ± 0.07 10[4]  >2.12 ± 0.11 >10.00 ± 0.07 1.70 ± 0.29 >10.31 ± 0.12      0.56 ±0.07 9.81 ± 0.26 Average 2.49 >9.84 1.54 9.22 −0.89 4.13 p value —<0.005 — <0.005 (tape vs. (n = 5) (n = 9) biopsy)^(c) ^(d)Subject ID andclinical score of SLS site in brackets; scoring is described inMaterials and Methods. ^(e)ΔC_(t) and minimum ΔC_(t) calculations aredescribed in Materials and Methods; an entry of “—” indicated thatinsufficient RNA was recovered to provide a meaningful estimate of theindicated ΔC_(t). A value preceded by “>” indicates that the mRNA forIL-1β or IL-8 was not detected in the sample, therefore therespectiveC_(t) was assigned a value of 37 and a minimum ΔC_(t) is given (i.e.ΔC_(t) = 37 − C_(t,actin)). ^(f)Two-way, paired t-test comparing ΔC_(t)for tape versus biopsy for a given skin treatment.

TABLE V Percentage of the measurement of gene expression level unchangedfor each of all possible pair-wise comparisons among GeneChips (A5, A6,A9, C1, C2, and C3) hybridized with aRNA obtained from three differentlocations on the upper back of two subjects (A and C). Gene Chip/SubjectID A5 A6 A9 C1 C2 C3 A5   100% A6 88.90%   100% A9 90.80% 86.10%   100%C1 89.80% 88.20% 87.40%   100% C2 85.00% 85.30% 83.10% 89.60%   100% C388.00% 88.00% 87.30% 88.90% 83.70% 100%

TABLE VI Functional grouping of top 100 differentially expressed genesbetween untreated and SLS treated conditions with p-values less than 1.4× 10⁻¹⁰ and PPDE(p) values greater than 0.99. Reference if KnownInvolvement in Injury/ Accession Number Gene Name Fold InflammationStructural proteins: X99142.1 Hair keratin, hHb6 −167.9 AJ406939.1Keratin associated protein 4.7 −51.9 (KRTAP4.7) BF740152 Myosin IE 26.2NM_000381.1 Midline 1 (OpitzBBB syndrome) −32.9 (MID1) NM_030966.1Keratin associated protein 1.3 −42.6 (KRTAP1.3) Z24727.1 Tropomyosin 1(alpha) −17.8 NM_002275.1 Keratin 15 (KRT15) −26.2 (Raval, Bharadwaj etal. 2003) NM_030975.1 Keratin associated protein 9.9 −859.3 (KRTAP9.9)Proteinases and protease inhibitors: L10343 Elafin/skin derived proteaseinhibitor 57.8 (Molhuizen and 3 (SKALP) Schalkwijk 1995) NM_002422.2Metalloproteinase 3 (Stromelysin 1) 109.9 (Pilcher, Wang et al. 1999;Fray, Dickinson et al. 2003) NM_003254.1 Tissue inhibitor ofmetalloproteinase 35.1 (Lobmann, Ambrosch et 1 (TIMP1) al. 2002)NM_001109.1 Disintegrin and metalloproteinase 97.1 (Kahari andSaarialhoKere domain 8 (ADAM8) 1997) NM_000362.2 Tissue inhibitor ofmetalloproteinase −29.7 (Lobmann, Ambrosch et 3 (TIMP3) al. 2002)U08839.1 Urokinase-type plasminogen 19.3 (Chung, Lee et al. 1996)activator receptor NM_004994.1 Matrix metalloproteinase 9 24.2 (Kahariand SaarialhoKere (gelatinase B) 1997; Herouy 2001) NM_001912.1Cathepsin L (CTSL) 18.5 (Kawada, Hara et al. 1997; Benavides, Starost etal. 2002; Welss, Sun et al. 2003) NM_000129.2 Coagulation factor XIII,A1 20.4 (Chung, Lee et al. 1996; polypeptide (F13A1) Ichinose 2001)NM_001150.1 Alanyl (membrane) aminopeptidase 35.3 (Lendeckel, Arndt etal. (aminopeptidase N/CD13) 2003) Cytokines, chemokines and theirreceptors: NM_002984.1 Small inducible cytokine A4 136.3 (Asadullah,Sterry et al. (SCYA4) 2002; Dong, McDermott et al. 2003) NM_003856.1Interleukin 1 receptor-like 1 89.4 (Asadullah, Sterry et al. (IL1RL1)2002; Dong, McDermott et al. 2003) AI421071 Chemokine (C—C motif)receptor 1 334.7 (Asadullah, Sterry et al. 2002; Dong, McDermott et al.2003) NM_002090.1 GRO3 oncogene 74.2 (Asadullah, Sterry et al. 2002;Dong, McDermott et al. 2003) NM_000640.1 Interleukin 13 receptor, alpha2 53.6 (Asadullah, Sterry et al. (IL13RA2) 2002; Dong, McDermott et al.2003) R64130 Pro-platelet basic protein 66.6 (Asadullah, Sterry et al.2002; Dong, McDermott et al. 2003) NM_001511.1 GRO1 oncogene (melanomagrowth 26.7 (Asadullah, Sterry et al. stimulating activity, alpha) 2002;Dong, McDermott et al. 2003) NM_001838.1 Chemokine (C—C motif) receptor7 25.5 (Asadullah, Sterry et al. (CCR7) 2002; Dong, McDermott et al.2003) NM_001558.1 Interleukin 10 receptor, alpha 171.1 (Asadullah,Sterry et al. (IL10RA) 2002; Dong, McDermott et al. 2003) NM_001562.1Interleukin 18 −13.8 (Asadullah, Sterry et al. 2002; Dong, McDermott etal. 2003) NM_006850.1 Suppression of tumorigenicity 16/Il- 65.7(Asadullah, Sterry et al. 24 2002; Dong, McDermott et al. 2003)NM_000576.1 Interleukin 1, beta (IL1B) 17.2 (Asadullah, Sterry et al.2002; Dong, McDermott et al. 2003) NM_006273.2 Small inducible cytokineA7 44 (Asadullah, Sterry et al. (SCYA7) 2002; Dong, McDermott et al.2003) Complement and complement receptors: NM_012072.2 Complementcomponent C1q 78.7 (Verhoef 1991; Bayon, receptor (C1QR) Alonso et al.1998) NM_001736.1 Complement component 5 receptor 1 121.5 (Verhoef 1991;Bayon, (C5R1) Alonso et al. 1998) U62027.1 Anaphylatoxin C3a receptor51.5 (Verhoef 1991; Bayon, Alonso et al. 1998) Histocompatibilitycomplex: NM_021983.2 Major histocompatibility complex, 26.7 (Alberts,Bray et al. 1994) class II, DR beta 4 (HLA-DRB4) AJ297586.1 MHC class IIantigen (HLA-DRB1 37.1 (Alberts, Bray et al. 1994) gene) X76775 Majorhistocompatibility complex, 42 (Alberts, Bray et al. 1994) class II, DMalpha (HLA-DMA) M27487.1 MHC class II DPw3-alpha-1 chain 32.8 (Alberts,Bray et al. 1994) M60334.1 MHC class II HLA-DR-alpha 23.7 (Alberts, Brayet al. 1994) NM_002118.1 Major histocompatibility complex, 18.3(Alberts, Bray et al. 1994) class II, DM beta (HLA-DMB) AA807056 Majorhistocompatibility complex, 24.4 (Alberts, Bray et al. 1994) class II,DR beta 3 AF005487.1 MHC class II antigen (DRB6) 22.4 (Alberts, Bray etal. 1994) Growth factors: NM_003862.1 Fibroblast growth factor 18(FGF18) −21.8 NM_013959.1 Neuregulin 1 (NRG1) 23.3 (Vermeer, Einwalteret al. 2003) Receptors and cell surface ligands: NM_013252.1 C-typelectin, superfamily member 5 75.4 (Kilpatrick 2002) (CLECSF5)NM_000560.1 CD53 antigen 94.6 (Alberts, Bray et al. 1994) Z22969.1 CD163antigen/M130 antigen 63.4 (Alberts, Bray et al. 1994) NM_018643.1Triggering receptor expressed on 55.2 (Colonna 2003) myeloid cells 1(TREM1) Y00062.1 CD45/T200 leukocyte common 61.9 (Alberts, Bray et al.1994) antigen NM_002438.1 Mannose receptor, C type 1 (MRC1) 69.8 (Baker,Ovigne et al. 2003) NM_004106.1 Fc fragment of IgE, high affinity I, 51(Alberts, Bray et al. 1994) receptor (FCER1G) NM_005849.1 Immunoglobulinsuperfamily, 44.5 (Alberts, Bray et al. 1994) member 6 (IGSF6)NM_004951.1 Epstein-Barr virus induced gene 2 34.9 (Alberts, Bray et al.1994) (lymphocyte-specific G protein- coupled receptor) (EBI2) BG236280CD86 antigen 23.9 (Alberts, Bray et al. 1994) AF313468.1 Dendriticcell-associated C-type 25.6 (Kilpatrick 2002) lectin-1 NM_003264.1Toll-like receptor 2 (TLR2) 28.5 (Alberts, Bray et al. 1994) NM_016184.1C-type lectin, superfamily member 6 19.9 (Kilpatrick 2002) (CLECSF6)NM_005211.1 Colony stimulating factor 1 receptor 37.4 (Alberts, Bray etal. 1994) NM_001828.3 Charcot-Leyden crystal 117.2 (Ackerman, Liu et al.protein/Galectin-10 2002) NM_002003.2 Ficolin 1 (FCN1) 17.6 (Alberts,Bray et al. 1994) M98399.1 CD36 81.7 (Alberts, Bray et al. 1994)Membrane transport: NM_022003.1 FXYD domain-containing ion −42.3transport regulator 6 (FXYD6) NM_006931.1 Solute carrier family 2(facilitated 48.3 glucose transporter), member 3 (SLC2A3) Intracellularsignal transduction: NM_005335.1 Cell-specific Lyn substrate 1 35.8(HCLS1) NM_003332.1 TYRO protein tyrosine kinase 219.3 (Lucas, Daniel etal. binding protein (TYROBP) 2002) NM_002463.1 Myxovirus (influenza)resistance 2 30.1 (Melen, Keskinen et al. 1996) AI123251 Lymphocytecytosolic protein 2 140.1 NM_002048.1 Growth arrest-specific 1 (GAS1)−76.6 AF183421.1 Small GTP-binding protein rab22b 22.9 AI356412 v-yes-1Yamaguchi sarcoma viral 27.3 related oncogene homolog AF039555.1Visinin-like protein 1 (VSNL1) −12.5 BC002671.1 Dual specificityphosphatase 4 41.8 NM_014380.1 p75NTR-associated cell death −8.1executor Enzymes: NM_003364.1 Uridine phosphorylase (UP) 46.3NM_002933.1 Ribonuclease, RNase A family, 1 86.7 (RNASE1) NM_005746.1Pre-B-cell colony-enhancing factor 35.5 (Samal, Sun et al. 1994) (PBEF)NM_000382.1 Aldehyde dehydrogenase 3 family, −17.2 member A2 (ALDH3A2)NM_021615.1 Carbohydrate (N-acetylglucosamine 68.3 6-O) sulfotransferase6 (CHST6) W46388 Superoxide dismutase 2, 15.6 mitochondria Extracellularmatrix associated proteins: NM_002727.1 Proteoglycan 1, secretorygranule 65.3 (PRG1) NM_004385.1 Chondroitin sulfate proteoglycan 2 30.8(Syrokou, Dobra et al. (versican) 2002) X77598.1 Laminin alpha 3 chain(LAM A3) 18.7 BF055462 Thrombospondin 1 55.5 (Vallejo, Mugge et al.2000) Transcription factors: AU145890 Forkhead box C1 −18.8 BC001283.1Nuclear factor IB −11.5 Others: AF245505.1 Adlican −65.4 U03891.2Phorbolin 1 52.7 NM_020987.1 Ankyrin 3, node of Ranvier (ankyrin −42.3G) (ANK3) NM_006762.1 Lysosomal-associated multispanning 41.2 membraneprotein-5 (LAPTM5) U56725.1 Heat shock 70 kD protein 2 −26.2 NM_001442.1Fatty acid binding protein 4, 29 adipocyte (FABP4), NM_014583.1 LIM andcysteine-rich domains 1 −25.4 (LMCD1) NM_015714.1 Putative lymphocyteG0G1 switch 52.8 gene (G0S2) NM_005410.1 Selenoprotein P (SEPP1) −16NM_002965.2 S100 calcium-binding protein A9 21.8 (Kerkhoff, Eue et al.(calgranulin B) 1999; Theory, Roth et al. 2001) BC006471.1 ALL1-fusedgene from chromosome −14.8 1q NM_006332.1 Interferon, gamma-inducibleprotein 31.4 (Phan, Lackman et al. 30 (IFI30) 2002) AF063606.1 Brainmy048 protein −63.1 NM_006851.1 Glioma pathogenesis-related protein 78.2(RTVP1) AA149745 Tripartite motif proteinTRIM2 −15.4

EXAMPLE 3

Non-invasive Isolation of Epidermal RNA from Psoriatic Patients UsingTape Stripping Method Provided herein

This example illustrates the isolation and detection of nucleic acidsfrom psoriatic lesions and the identification of genes whose expressionis associated with psoriatic lesions. This example summarizes theresults of tape harvesting lesional and non-lesional skin in 24psoriatic patients in various treatment stages. The goal of thisinvestigational work was to determine if DermTech's Epidermal GeneticInformation Retrieval Technology (EGIR), which is a tape disk used witha synthetic rubber-based adhesive (Adhesive Research, Glen Rock, Pa.) ona polyurethane film (Product No. 90068), could successfully recover RNAfrom the surface of lesional and non-involved skin from psoriaticpatients; and to semi-quantitate recovered RNA for specific mRNAmolecules known to be elevated in psoriatic lesions. The data generatedfrom these patients demonstrates that RNA can be recovered and thatmRNAs for TNFα, IFNγ, CD2, GAPDH, and β-actin can be detected andsemi-quantitated in tape harvested epidermal samples. Nanogramquantities of RNA were recovered from 92% of tape harvested psoriaticplaques. Recovery of RNA from non-involved control skin was lesssuccessful with a 31% success rate. The recovery of RNA fromnon-lesional skin was not random because some subjects could be tapeharvested with repeated success while others could not. The recovery ofRNA from non-lesional psoriatic skin contrasts with the success of tapeharvesting normal skin of healthy individuals, which has an 85% successrate. Semi-quantitative RT-PCR analysis demonstrated that at least 6patients had significantly elevated TNFα mRNA levels in psoriaticplaques, 4 patients had elevated IFNγ mRNA and 3 had increased CD2message relative to β-actin. 18 patients could not have the relativechange of any marker assayed in psoriatic lesions because ofinsufficient RNA collection from control skin. However, analysis ofΔC_(t) values in 21 patient's lesions demonstrated a highly significantdifference between TNFα and CD2 mRNA levels relative to β-actin inpsoriatic versus control skin. These data suggest that TNFα and CD2 mRNAwere in fact elevated in most patients and that patients could becategorized into two groups, those with elevated TNFα and CD2 mRNA andthose with elevated TNFα, CD2 and IFNγ mRNAs. The data demonstrate thatthere are distinct relative abundances of the 3 mRNAs with respect toβ-actin in psoriatic versus non-lesional skin, differences which arecommon across subjects. These preliminary results are very encouragingand demand confirmation with additional patients.

Materials and Methods

Clinical: Sample collection was done at the University of Utah incollaboration with Dr. Gerald Krueger. For each body site a total of 4fresh tapes were sequentially applied once to a single site and removed.Tapes were put into individual eppendorf tubes, frozen at −80 andexpress mailed on dry ice to DermTech International where all subsequentanalysis was performed. The tape used for tape stripping was a syntheticrubber-based adhesive MA70 (Adhesive Research) on a polyurethane film.

Isolation of RNA: Total RNA was isolated using our standard method andthe RNeasy fibrous tissue kit (Qiagen). The 4 tapes used to harvest eachsite were combined in buffer RLT and extracted together according to themanufacturer's guidelines using our standard adaptations.

Quantitation of RNA mass: Samples were quantified by non-competitivesemi-quantitative RT-PCR using a fluorescence-based 5′-nuclease assay(“Real-time” PCR) on an ABI 7000 or 7900. Each sample was reversetranscribed in triplicate and each cDNA amplified and quantified induplicate; the resulting 6 C_(t) values were converted to RNA masses,which were averaged to yield the data in Table 1. C_(t) values wereconverted to RNA masses using the standard curve method. A standardcurve was generated with total RNA from human spleen. The accuracy ofthis method assumes that the relative amount of β-actin in human spleenRNA is identical to that in skin samples recovered by tape stripping.All assays were performed using Predeveloped Assay Reagents purchasedfrom Applied Biosystems. The data from the first 11 subjects (SampleSets 1 and 2) was gathered using multiplex assays (same tube assay ofactin and the mRNA of interest). Thereafter, all analyses have been donein a single tube single analysis format. All ΔC_(t) and ΔΔC_(t)calculations are done with C_(t) values determined during the sameexperiment (i.e. simultaneous amplification/detection).

Semi-quantitation of mRNA levels: Messenger RNA (mRNA) levels weresemi-quantified for GAPDH, TNFα, IFNγ, and CD2 using non-competitiveRT-PCR and the comparative (ΔΔC_(t) method; 5′-nuclease assay). In theΔΔC_(t) method individual mRNAs (“mRNA_(x)” i.e. the RNA-of-interest)are semi-quantified by normalization to β-actin mRNA (mRNA_(a)) and thisratio is divided by the similar ratio from an uninvolved skin site, astep referred to as “calibration”. The resulting number is an indicationof the change of (mRNA_(x)/mRNA_(a)) in lesional versus non-lesionalskin.

Background of the ΔΔC_(t) method (Parts of the following discussion andadditional information not discussed here can be found in ABI UserBulletin #2, which can be found at:http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf): Duringamplification of a sample using fluorescence detection and the5′-nuclease assay, the net (background corrected) fluorescence of asample is directly related to the amount of PCR product synthesized,which is related to the initial amount of specific mRNA in the sample.This fluorescence, called ΔR_(n), is related to the amount of PCRproduct by the equation:ΔR_(n)∝X_(T)=X₀2^(C)  1]where X_(T) is the amount of total PCR product at cycle C and X₀ is theinitial amount of mRNA_(x). During the PCR reaction, ΔR_(n) risesexponentially (under non-competitive conditions in the early stages ofthe reaction); when ΔR_(n) rises significantly above background, it issaid to have reached a threshold value and equation 1] becomes:ΔR_(N,x)=K_(x)X₀2^(Ct,x)  2]where K_(x) is a spectroscopic constant specific to the fluorescentprobe and the reaction conditions and C_(t,x) (the threshold value) isthe number of PCR cycles required to reach the threshold fluorescenceΔR_(n). A similar equation can be written for the normalization mRNA,which in this case is β-actin:ΔR_(N,A)=K_(A)A₀2^(Ct,A)  3]where K_(A) is an actin specific constant and A₀ is the initial numberof actin mRNA molecules. By dividing equation 2 by 3 and rearranging, weobtain an equation relating the fraction of mRNA_(x) to β-actin mRNA inour unknown or “experimental” sample:

$\left. 4 \right\rbrack\mspace{14mu}\left( {\frac{X_{0}}{A_{0}} = {\frac{\Delta\; R_{n,X}}{\Delta\; R_{n,A}}K_{AX}2^{{- \Delta}\; C_{T,{Exp}}}}} \right)_{Exp}$where K_(AX)=K_(A)/K_(X) and ΔC_(t,Exp)=C_(t,X)−C_(t,A). This equationrelates the initial (unknown) number of mRNA molecules to theexperimentally determined threshold cycle number. From the equation, wecan see that the ratio of the two mRNAs is not only a function of theexperimentally derived C_(t) values but also a function of the constantK_(AX) (an unknown), and the two ΔR_(n) values, which are determined andreported by the instrument. Thus without knowledge of K_(AX), thecomparative method does not reveal the absolute ratio of two mRNAs in asingle sample. However, by writing a similar equation for a second“calibrator” sample

$\left. 5 \right\rbrack\mspace{14mu}\left( {\frac{X_{o}^{Cal}}{A_{o}^{Cal}} = {\frac{\Delta\; R_{n,X}^{Cal}}{\Delta\; R_{n,A}^{Cal}}K_{AX}2^{{- \Delta}\; C_{T}^{Cal}}}} \right)_{Cal}$and dividing equation 4 by 5 we obtain:

${\left. 6 \right\rbrack\mspace{14mu}{\frac{X_{0}}{A_{0}}/\frac{X_{0}^{Cal}}{A_{0}^{Cal}}}} = {\left( \frac{\Delta\; R_{n,X}}{\Delta\; R_{n,A}} \right)K_{AX}{2^{{- \Delta}\; C_{t,{Exp}}}/\left( \frac{\Delta\; R_{n,X}^{Cal}}{\Delta\; R_{n,A}^{Cal}} \right)}K_{AX}2^{{- \Delta}\; C_{t,{Cal}}}}$If the experimental and calibrator samples are analyzed during the sameexperiment, the threshold values are equal

Δ R_(n, x) = Δ R_(n, x)^(Cal)  and  Δ R_(n, A) = Δ R_(n, A)^(Cal),and because K_(AX) is identical for both samples, equation 6 simplifiesto:

${\left. 7 \right\rbrack\mspace{14mu}\left( \frac{X_{0}}{A_{0}} \right)\left( \frac{A_{0}^{Cal}}{X_{0}^{Cal}} \right)} = 2^{{- {\Delta\Delta}}\; C_{t}}$where ΔΔC_(t)=ΔC_(t, exp)−ΔC_(t, cal), recalling that ΔC_(t, exp) isC_(t,x)−C_(t,actin) for the experimental sample and ΔC_(t,cal) is theanalogous difference for the calibrator sample. Thus equation 7 allowsus to infer a change of mRNA_(x)/mRNA_(a) between the sample of interest(psoriatic tissue) and a calibrator sample (non-lesional skin) directlyfrom C_(t) measurements. Note that if one adds the further assumptionthat the “housekeeping” gene does not change its expression relative tototal RNA then equation 7 simplifies to:

${\left. 8 \right\rbrack\mspace{14mu}\left( \frac{X_{0}}{X_{0}^{Cal}} \right)} = 2^{{- {\Delta\Delta}}\; C_{t}}$It should be noted that this final simplification is not necessary todraw significance from C_(t) values for diagnostic purposes.

Analysis of ΔC_(t) values without calibration: The ΔΔC_(t) methodrequires the use of a calibrator sample to eliminate unknown constantsin Equation 4 and relate the ΔC_(t) values directly to mRNA levels inthe two samples. It would be advantageous to be able to analyze ΔC_(t)values without the necessity of a calibrator sample. Equation 4 seems tooffer several means of doing this. Equation 4 relates X₀/A₀ to ΔC_(t);if one could experimentally change this ratio (for instance by using invitro transcribed RNA) then a graph of ΔC_(t) vs. log [X₀/A₀] shouldhave a slope related to the known ΔR_(n)'s and the unknown K_(AX), whichcould be solved for K_(AX), which in turn could be used to directlyrelate ΔC_(t) to mRNA_(x)/mRNA_(a) in a sample without calibration.

An alternative strategy is to inspect equation 4 for the variables whichcontribute to the observed ΔC_(t) and assess their contributions to it.By taking the log of equation 4 we obtain:

$\left. 9 \right\rbrack\mspace{14mu}\begin{matrix}{{\log\left( \frac{X_{0}}{A_{0}} \right)} = {\log\left( {\frac{\Delta\; R_{n,X}}{\Delta\; R_{n,A}}K_{AX}2^{{- \Delta}\; C_{T,{Exp}}}} \right)}} \\{= {{\log\left( \frac{\Delta\; R_{n,X}}{\Delta\; R_{n,A}} \right)} + {\log\mspace{14mu} K_{AX}} + {\log\mspace{14mu} 2^{{- \Delta}\; C_{T,{Exp}}}}}} \\{= {{\log\left( \frac{\Delta\; R_{n,X}}{\Delta\; R_{n,A}} \right)} + {\log\mspace{14mu} K_{AX}} - {\Delta\; C_{T,{Exp}}\log\mspace{14mu} 2}}}\end{matrix}$solving equation 9 for ΔC_(t) yields

${\left. 10 \right\rbrack\mspace{14mu}\Delta\; C_{T,{Exp}}} = \frac{{- {\log\left( \frac{X_{0}}{A_{0}} \right)}} + {\log\left( \frac{\Delta\; R_{n,X}}{\Delta\; R_{n,A}} \right)} + {\log\mspace{14mu} K_{AX}}}{\log\mspace{14mu} 2}$We can see that ΔC_(t,Exp) depends on 3 factors; X₀/A₀;ΔR_(n,X)/ΔR_(n,A); and K_(AX). If we compare two samples analyzed duringthe same experiment, the ΔR_(n) values will be identical and cannotcontribute to changes in the ΔC_(t). Likewise, since the K_(AX) valuesare identical we can see that ΔC_(t) values will only change if X₀/A₀changes. Therefore, under same plate measurements, it is valid tocompare ΔC_(t) values without a calibration step. A logical extension isto ask if ΔC_(t) measurements from separate experiments can be compared.In such an event it is likely that ΔR_(n)'s will be different, howeverequation 10 allows one to calculate an adjusted ΔC_(t) that will reflectthe effect of differing ΔR_(n) values. Rearrangement of equation 10gives:

${{\left. 11 \right\rbrack\mspace{14mu}\Delta\; C_{T,{Exp}}} - \frac{\log\left( \frac{\Delta\; R_{n,X}}{\Delta\; R_{n,A}} \right)}{\log\mspace{14mu} 2}} = \frac{{- {\log\left( \frac{X_{0}}{A_{0}} \right)}} + {\log\mspace{14mu} K_{AX}}}{\log\mspace{14mu} 2}$The expression to the left of the equality in equation 11 is an“adjusted” ΔC_(t) which accounts for differing ΔR_(n)'s. We can see thatthe adjusted ΔC_(t) only depends on A₀/X₀ and K_(AX). In this caseK_(AX) will be constant for both samples—given identical reactionconditions and probes—and any differences between adjusted ΔC_(t)'s willbe a consequence of differences in X₀/A₀.

Data handling and statistical analysis: For each mRNA assay we have 6replicate measurements of the C_(t). The average of these C_(t) valuesis used to calculate the ΔC_(t,x) for the sample and calibrator. It isour experience that as C_(t) values approach 37 cycles, individualassays can produce undetectable readings or C_(t) values>37 with highvariability. In order to deal with combinations of undetectable andreadings greater than 37 with high standard deviations, we have adoptedthe following data management rules. Rule 1] if there are 4 or morereadings (out of 6 replicates) of undetectable, a C_(t) value of 37 (ourlimit of detection) is assigned to the sample; Rule 2] if an averageC_(t) is greater than or equal to 37 and the standard deviation isgreater than or equal to 1, a value of C_(t)=37 is assigned to the mRNAbeing measured. This last rule simply states that if an mRNA is at ourlimit of detection or reliable quantitation, we simply define it asundetectable in order to estimate its maximum value. Calculationsperformed with C_(t)≡37, where that value has been assigned by the aboverules, are indicated by enclosing the resulting data in parentheses.Outlier measurements are defined as outside the average of remainingmeasurements ±3 standard deviations. If a single measurement liesoutside this boundary it is eliminated from the calculations. AnmRNA_(x)/mRNA_(a) ratio is considered statistically different than thecontrol value (at 95% confidence) if the fold-change given by2^(−ΔΔCt±2std dev) does not overlap the control range given by2^(±2std dev).

Results

RNA Yield

Table 1 shows the yields of total RNA from non-lesional and lesionalskin in 24 patients. It is our experience with tape harvesting thatyields of less than 200 picograms are not useful for quantitating mRNAlevels 8-fold less abundant than β-actin. By applying this standard ofat least 200 picograms, we can categorize RNA recovery as successful ornot successful. Table 2 summarizes the results of categorizing the massdata by the 200-picogram criterion. We can see that tape harvestinglesional skin was very successful with 91% of samples having sufficientRNA for analysis. However, tape harvesting of non-lesional skin was lesssuccessful (31% success).

Because analysis of the second sample set suggested that non-lesionalskin might present recovery problems, subsequent sample sets included 2non-lesional skin sites as controls. A total of 12 patients each had 2control sites tape harvested. Of these 12 pairs of sites, 9 pairsproduced insufficient RNA and 3 pairs produced nanogram quantities ofRNA (Table 1). There were no pairs of mixed sites. This data stronglysuggests that the recovery of RNA from uninvolved skin is not a randomoccurrence but that some patients may in fact yield less RNA. A similarobservation applies to the skin of subjects with no psoriasis. However,the frequency of successful recovery from healthy subjects' normal skinis 84%. This contrasts quite strongly with the 31% recovery fromnon-lesional psoriatic skin. We conclude that 1] the uninvolved skin ofpsoriatic patients is different at the level of ability to recover RNAfrom the surface of the skin; and 2] that these 12 psoriatic patientscan be divided into two categories based on the ability to recover RNAfrom non-lesional skin. A caveat to this second conclusion is that bodylocation may be an important factor and the particular sites harvestedhave not yet been reported. It will be of interest to understand thedifferences between psoriatic patients who either do or do not yield RNAfrom non-lesional skin using EGIR adhesive films.

Relative Cytokine Levels

Table 3 reveals the relative increases in mRNAs for TNFα, IFNγ, and CD2in lesional compared to non-lesional skin. Table 3 shows that 6 patientshad significantly elevated TNFα/β-actin mRNA ratios compared tonon-lesional skin. In 15 patients we were unable to classify theTNF/actin mRNA ratio as elevated or not because of poor RNA recoveryfrom the control site.

The data for IFNγ shows 3 patients having elevated IFNγ/actin mRNAratios and 18 patients having no conclusion because of inadequatecontrol data. Data for CD2 reveals that 3 patients had elevatedCD2/actin mRNA levels, 2 had normal or borderline elevated levels(possible 2.4-fold increases) and 16 samples were indeterminate.

Discussion

In this work we have analyzed the results of tape harvesting psoriaticand non-lesional skin on 24 patients. We have quantified the RNArecovered and shown that when sufficient RNA is recovered, it can beproductively analyzed. We have found that RNA can be recovered with highfrequency from psoriatic plaques, while RNA is recovered with belowaverage frequency (compared to healthy subjects) from non-lesional skin.While it seems clear that recovery of RNA from non-lesional skin ispatient specific, we have not eliminated the possibility that body siteplays a major role in RNA recovery. The inability to efficiently collectnon-lesional skin data from psoriatic patients would seem to be anobstacle to the analysis of lesions (Table 3). However, the ultimategoal of this work is to devise an assay which requires only a lesionsample and no control skin (discussed below). Because such a goalrequires a foundational database of ΔC_(t) values from non-lesionalskin, or at the very least normal skin, we will continue to pursue ideasto increase the isolation of RNA from non-lesional skin.

Several strategies can possibly be used for increasing the yield of RNAfrom non-lesional skin sites on, psoriatic patients or devising a methodof calibrating ΔC_(t)'s from lesions without using non-lesionalcontrols. These strategies are:

-   -   1. Optimization of tape usage: It is probable that different        techniques of applying tape to the skin could affect the        recovery of RNA. In particular, aggressive application to the        skin is necessary. Two physicians have been trained in the        method. The consensus was that the training was highly        instructive and will make a difference in how efficiently        non-lesional skin is sampled.    -   2. In principle, only one non-lesional (i.e. unaffected) control        is needed, since that data could be used to calibrate lesional        samples taken at later time points. Thus a non-lesional sample        could involve the one time use of up to 10 applications of tape        to a single site to insure obtaining a sample.    -   3. There is anecdotal evidence that body location is a        significant factor for RNA recovery. This hypothesis is being        tested in healthy individuals. Preliminary data suggests that        the upper arm, over the deltoid, the upper back over the        scapular spine and the periauricular (mastoid) region may be        superior RNA yielding sites.    -   4. A single shave biopsy could act as a control for all        subsequent analysis.    -   5. It is possible, perhaps desirable to use commercially        available RNA as a “universal” calibrator.

It is relatively common in medical diagnostics to analyze tissue samplesand draw conclusions—without reference to a control sample—based onpopulation data. For instance, blood is routinely analyzed andconclusions based on whether the value of a given parameter falls withina normal or abnormal range. In this example there is no “control”sample, the patient only has one blood source; the control is based onthe range of values found in normal samples. Likewise, we should be ableto evaluate psoriatic plaques without reference to a control sample fromthat individual. While the most obvious candidate for a diagnosticparameter is the mRNA_(x)/reference mRNA ratio, we have seen thatwithout absolute quantification of mRNA, we cannot use C_(t) values tocalculate the absolute ratio of 2 mRNAs. This does not mean that wecannot use uncalibrated ΔC_(t)'s to compare and classify lesions.

When the ΔΔC_(t) method is used to semi-quantitate mRNA a calibratorsample is required that allows the cancellation of unknown constants andconsequent direct relation of ΔΔC_(t) to relative mRNA_(x)/mRNA_(a)levels in the unknown and calibrator samples (formula 6; Materials andMethods Section). If one were to compare ΔC_(t) values amongst samples(i.e. an uncalibrated sample comparison), it would be necessary toaccount for the variables other than X₀/A₀ that contribute to theobserved ΔC_(t).

Equation 4 relates the ΔC_(t) value to the mRNA_(x)/mRNA_(a) ratio inthe sample. Rearrangement of equation 4 (Materials and Methods) showsthat ΔC_(t) is a function of 1] the mRNA_(x)/mRNA_(a) ratio in thesample; 2] the ΔR_(n) values for the message of interest andnormalization mRNA; and 3] the spectroscopic constant K_(AX). If thesamples to be compared are assayed during the same experiment, then theΔR_(n) values will be identical and will not contribute to anydifferences in ΔC_(t). The use of the same sample volumes, and probeswill likewise assure that K_(AX) is the same for all samples. Therefore,it should be possible to compare uncalibrated ΔC_(t) values—knowing thatthey are only a function of X₀/A₀—and evaluate the hypothesis that themRNA_(x)/mRNA_(a) ratio is different in psoriatic versus non-psoriaticskin and that this difference extends across individuals in apopulation.

The data in Table 4 can be used to test this hypothesis. Table 4contains the ΔC_(t) values for all samples. Under each mRNA-of-interest,the ΔC_(t) values for lesions are sorted into one of three columns. Thefirst column on the left (signified by ⇑) contains ΔC_(t) values fromlesion samples with significant increases in the mRNA_(x)/mRNA_(a) ratiocompared to control skin (Table 3). The second column contains ΔC_(t)values from lesions with no significant change from control skin. Thethird column contains ΔC_(t) values from unclassified lesions. Thetypical reason for a lesion being unclassified is due to insufficientRNA collection from the control sample (non-lesional skin). A fourthcolumn contains ΔC_(t) values from non-lesional skin.

By sorting ΔC_(t) values into known and unknown categories, we can usestatistical tests to determine if the categories define unique ranges.In Table 4 the average ΔC_(t,TNF) in samples with increased TNFα mRNAlevels is 5.18. In contrast, the average ΔC_(t,TNF) for control skinsamples is 9.53. A t-test shows that the difference between ΔC_(t)values for lesional vs. non-lesional skin is highly significant(p=0.0007). A similar analysis done on the unclassified ΔC_(t,TNF) datashow that the unclassified lesion samples have ΔC_(t,TNF) valuessignificantly different than control values (Table 4; p<0.0005). Thus itappears that the unclassified lesion samples consist largely of sampleswith elevated TNFα/β-actin mRNA ratios.

The same analysis can be applied to the ΔC_(t,CD2) data. Table 4 showsthat the ΔC_(t,CD2) values for samples with elevated CD2 mRNA issignificantly different than control samples (p=0.001) and thatΔC_(t,CD2) values for unclassified lesion samples are also differentthan control values (p<0.005). It therefore appears that ourunclassified lesional samples represent mostly samples with increasedCD2 mRNA levels.

Similar analysis of IFNγ mRNA reveals that ΔC_(t,IFN) in lesions withelevated IFNγ/actin mRNA ratios have significantly different ΔC_(t,IFN)values than control skin (Table 4; p=0.02). When the ΔC_(t,IFN) valuesfor unclassified lesion samples are compared to control skin, there isnot as high a confidence that they are different than non-lesional skin(p=0.12). Our interpretation of this data is that the unclassifiedcategory contains a mixture of samples, some of which have elevated IFNγlevels and some of which do not. Indeed, inspection of the unclassifiedΔC_(t,IFN) values shows a range of values from a low of 2.01 (almostcertainly abnormally high) to a high of 11.14 (probably normal). Fromthis data we can conclude that most of the samples have elevated TNFαand CD2 mRNA levels and that some proportion of patients have elevatedIFNγ mRNA levels. This suggests that we have 2 classes of patients,those with high levels of all 3 markers and those with high levels ofTNF and CD2 and normal levels of IFNγ. It is of high interest to definethe clinical differences that create these categories:

The above analysis of ΔC_(t) values establishes the likelihood thatthere is a range of values for lesional psoriatic skin and non-lesionalskin unique to the skin type (Table 4). The heterogeneity in theunclassified ΔC_(t,IFNγ) data highlights the larger question of how tocategorize lesional ΔC_(t) data for which we have no control samples. Itis clear that in order to classify ΔC_(t)'s we need a database ofcalibrated ΔC_(t) values from lesional and non-lesional (or normal skinfrom healthy individuals) skin. At our current rate of success obtainingRNA from non-lesional skin, we will need approximately 100 patients toget 25–30 calibrated ΔC_(t)'s from which we can define normal andabnormal ranges. The fact that a subset of patients seems to be good RNAyielders raises an interesting question. If in fact these individualshave “different” normal skin then they may not be appropriate controls(for some markers, which by definition would be interesting). In fact,normal healthy people may be the best control group.

TABLE 1 Total RNA yield summary. Skin Type and Yield* Non-lesionalLesional ID Yield Std dev Yield Std dev  1 (W.S)¹ 5.1 1.2 44 8  2(J.C.)¹ 4.6 0.8 11 2.9  3 (J.S.)¹ — 1.7 0.49  4 (D.C.)¹ 29 9.1 18 1.8  5(J.P.)¹ 138 2.7 1.2 0.45  1² 0.031 0.011 8.6 1.4  2² 0.045 0.016 12.61.5  3² 0.15 0.017 5.12 0.58  4² 3.94 0.63 0.053 0.006  5² 0.0063 0.850.13  6^(2,3) ND ND  60 (70)⁴ 0.02 0.018 8.1 0.86 100 (105)⁴ 0.16 0.04418 1.2 100 (110)⁴ 0.027 0.025 115 (120)⁴ 0.027 0.012 14 1.2 115 (125)⁴0.099 0.027 130 (135)⁴ 18 1.6 40 4.9 130 (140)⁴ 38 3.9 150 (160)⁴ 12 1.29.3 0.66 150 (170)⁴ 1.8 0.26 180 (190)⁵ 0.053 0.018 11.6 0.7 180 (200)⁵ND 210 (220)⁵ ND 2.32 0.06 210 (230)⁵ ND 250 (260)⁵ ND 0.31 0.04 250(260)⁵ ND 270 (280)⁵ 0.017 0.024 1.37 0.08 270 (290)⁵ ND 300 (310)⁵ 0.020.015 1.4 0.12 300 (320)⁵ ND 340 (350)⁵ 0.022 0.021 5.92 0.64 340 (360)⁵0.09 0.029 370 (380)⁵ 24.5 1.3 15.3 2.82 370 (390)⁵ 7.33 0.44 400^(5,6)0.027 0.024 410^(5,6) ND 420^(5,6) 0.1 0.059 *Yield reported innanograms total RNA; ND = none detected ¹University of Utah Round 1²University of Utah Round 2 ³Subject consented to one tape application⁴University of Utah Round 3; one patient had a single control (sample ID70) and lesion (ID 60) sampled, the remaining 4 patients had 2 controlsand 1 lesion sampled; the control ID is in parenthesis ⁵University ofUtah Round 4; 2 controls and 1 lesion sampled; control ID is inparenthesis ⁶These samples have not been assigned sample/lesiondesignations and may have been transported on ice an undefined amount oftime before freezing

TABLE 2 Summary of mass recovery in sample sets 1–4. Lesional¹ Control¹Patient Total 21/23 (91%) 11/35 (31%) 24 ¹The fraction of samples with≧200 picograms RNA; from a total of 23 patients, subject 6 from Round 2is not included in these data (but is included in the patient total).

TABLE 3 Relative levels of TNFα, IFNγ and CD2 in psoriatic lesions.Relative mRNA/β-actin mRNA levels¹ Sample# TNFα IFNγ CD2  1²  (3.23) (0.06)  (0.83)  2²   10** ND  (1.9)  3² — — —  4²   8.9** ND   3.9  5²  42** (513)**  20**  1³ — — —  2³ — — —  3³ — — —  4³ — — —  5³ — — — 60⁴ — — — 100⁴ — — — 115⁴ — — — 130⁴   11.3**  (1.7)   2.38**   8.3** (4.3)**   2.42 150⁴  (37)**  558**  19**  (11)**  (5.7)** 180⁵ — — —210⁵ — — — 250⁵ — — — 270⁵ — — — 300⁵ — — — 340⁵ — — — 370⁵  331**  38**  11.1** 400^(5,6) — — — ¹The fold-induction relative touninvolved skin is shown; Numbers in parentheses are lower limitestimates calculated by assigning a C_(t) = 37 to the mRNA of interest(TNFα, IFNγ or CD2), this estimate is used because the mRNA of interestwas not detectable in the control site;values with ** are consideredstatistically different than the control site at the 95% confidenceinterval as described in Materials and Methods; a “—” indicates thatinsufficient RNA was recovered from the control (typical) or lesionalsite;ND indicates no mRNA for gene of interest detected in either lesionor control sample (but RNA present); Samples 130 and 150 have 2different fold-increases corresponding to calibration to two differentnon-lesional skin sites. ²University of Utah Round 1 ³University of UtahRound 2 ⁴University of Utah Round 3; 1 patient had a single control andlesion sampled, the remaining 4 had 2 controls and 1 lesion sampled; thecontrol ID is in parenthesis ⁵University of Utah Round 4; 2 controls and1 lesion sampled; control ID is in parenthesis ⁶These samples have notbeen assigned sample/lesion ID and were transported on ice an undefinedamount of time before freezing

TABLE 4 Categorization and comparison of ΔC_(t) values. ΔCt (C_(t,gene)− C_(t,actin))¹ TNFα IFNγ CD2 Psoriatic Psoriatic Psoriatic ID ↑

UK Control ↑

UK Control ↑

UK Control  1 (W.S.)² 6.45 (8.14) 11.16 7.12 8.17 (7.89)  2 (J.C.)² 5.288.59 (8.21) (7.6) 6.91 (7.84)  3 (J.S.)² 5.04 NRNA 4.25 NRNA 4.64 NRNA 4 (D.C.)² 7.84 10.99 (8.32) (9.43) 7.59 9.54  5 (J.P.)² 4.41 9.81 2.96(11.96) 4.47 8.81  1³ 4.1 NRNA (8.19) NRNA 7.8 NRNA  2³ 2.84 NRNA 9.19NRNA 5.25 NRNA  3³ 4.02 NRNA 7.96 NRNA 5.58 NRNA  4³ NRNA 6.79 NRNA 9.91NRNA 6.84  5³ 3.07 NRNA (8.24) NRNA (5.33) NRNA  60 (70)⁴ 8.29 NRNA 9.98NRNA 7.72 NRNA 100 (105)⁴ 6.22 NRNA 10.09 NRNA 7.06 NRNA 100 (110)⁴ NRNANRNA NRNA 115 (120)⁴ 5.07 NRNA 11.14 NRNA 8.5 NRNA 115 (125)⁴ NRNA NRNANRNA 130 (135)⁴ 7.11 10.6 10.2 (10.96) 8.67 9.92 130 (140)⁴ 10.16 (12.3)9.94 150 (160)⁴ 5.13 (10.35) 3.49 12.62 4.48 8.72 150 (170)⁴ (6.99)(6.99) (6.99) 180 (190)⁵ 4.03 NRNA 4.68 NRNA 4.23 NRNA 180 (200)⁵ NRNANRNA NRNA 210 (220)⁵ 6.86 NRNA (8.18) NRNA 7.8 NRNA 210 (230)⁵ NRNA NRNANRNA 250 (240)⁵ 3.91 NRNA (4.98) NRNA (4.98) NRNA 250 (260)⁵ NRNA NRNANRNA 270 (280)⁵ 3.7 NRNA 3.47 NRNA 5.54 NRNA 270 (290)⁵ NRNA NRNA NRNA300 (310)⁵ 3.23 NRNA 2.01 NRNA 4.84 NRNA 300 (320)⁵ NRNA NRNA NRNA 340(350)⁵ 2.65 NRNA 4.6 NRNA 5.51 NRNA 340 (360)⁵ NRNA NRNA NRNA 370 (380)⁵1.28 9.66 6.67 11.93 6.63 10.09 370 (390)⁵ 9.64 (10.13) (10.13)400^(5,6) NRNA NRNA NRNA 410^(5,6) NRNA NRNA NRNA 420^(5,6) NRNA NRNANRNA Average* 5.18 4.63 9.53 4.37 7.39 10.40 5.19 8.13 6.4 9.12 p-value†0.0007 <0.0005 0.02 0.12 0.001 0.3 <0.005 ¹The ΔC_(t) value is definedas the C_(t) value for the mRNA of interest minus the C_(t) value forβ-actin (C_(t,mRNA) − C_(t,actin)). In some samples the mRNA of interestcannot be detected, in which case the C_(t) is defined as 37 cycles, ourlimit of detection; in such cases an estimated ΔC_(t) is calculated fromthe formula 37 − C_(t,actin) and is reported in parenthesis. A columnheaded by a ↑ contains data from lesions with statistically elevated(95% confidence interval) cytokine levels; a column headed with

contains data from lesions showing no significant change; a columnheaded by UK contains unclassified data (calibrator unavailable); NRNAindicates insufficient RNA recovered. ²University of Utah Round 1.³University of Utah Round 2. ⁴University of Utah Round 3; 1 patient hada single control and lesion sampled, the remaining 4 had 2 controls and1 lesion sampled; the control ID is in parenthesis. ⁵University of UtahRound 4; 2 controls and 1 lesion sampled; control ID is in parenthesis.⁶These samples have not been assigned sample/lesion ID and may have beentransported on ice an undefined amount of time before freezing.*Averages and standard deviations do not include estimated data (# inparentheses). †Two tailed t-test compared to non-lesional skin; ΔC_(t)values in parentheses are not included in the calculation.

EXAMPLE 4

Relative levels of mRNA as indicated by ΔC_(t) values in lesional andnon-lesional skin of psoriatic patients

This example illustrates the use of the tape stripping method disclosedherein and ΔC_(t) values, to characterize genomic expression in thestratum corneum of psoriasis lesional and non-lesional skin. Morespecifically, this study determines if ΔC_(t) values for various mRNAsknown to be upregulated in psoriatic lesions could be characterizedusing RNA recovered by tape stripping.

Methods

The tape stripping procedure and tape are identical to those disclosedin Example 3. One lesion was sampled and 3 independent uninvolved skin(UIS) sites were sampled per patient. The 3 uninvolved skin site sampleswere combined to produce one “global” control sample. Each site wassampled with 4 individual tapes, each sequentially applied and removedonce. mRNA was semi-quantitated using the comparative or ΔC_(t) methodusing β-actin as the normalizing message.

Results and Discussion

In this tape stripping study a total of 72 subjects lesions and 163normal skin sites were sampled. Some patients with less severe diseasewere only sampled on uninvolved skin sites. Each sample wassemi-quantitated for GAPDH, TNFα, IFNγ, CD2, Krt-16, IL-12B, and IL-23AmRNA which were normalized to β-actin mRNA. The results of these assaysare shown in Table 9.

TABLE 9 Population average ΔC_(t) values of select biomarker mRNAs inlesional and uninvolved skin of psoriatic patients Biomarker mRNA andAverage ΔC_(t) ^(b) GAPDH TNFα IFNγ Krt-16 CD2 IL-12B IL-23A Site^(a)ΔC_(t) SEM ΔC_(t) SEM ΔC_(t) SEM ΔC_(t) SEM ΔC_(t) SEM ΔC_(t) SEM ΔC_(t)SEM UIS 2.07 0.06 8.32 0.16 10.5  0.72 −1.33 0.12 8.43 0.3  11.5  1.8 9.03 0.63 (N) (163) (97)  (9) (137) (34)  (3) (10) Lesion 0.72 0.19 4.870.21  7.79 0.35 −2.57 0.23 6.56 0.18  8.55 0.28 5.7  0.31 (N)  (72) (72)(66)  (45) (69) (44) (45) ^(a)Tape stripped site, lesional or uninvolvedskin (UIS); the number of observations is listed below in parenthesis.^(b)ΔC_(t) is defined as C_(t,mRNAx) − C_(t,actin mRNA) where C_(t) isthe respective number of PCR cycles required to achieve thresholdfluorescence. In some samples the threshold could not be determined(i.e. the mRNA was not detectable) or was not assayed, thus the numberof observations is different for each mRNA. SEM is standard error of themean calculated as standard deviation divided by N^(1/2).

The data in Table 9 clearly demonstrates that there are differentrelative levels of all the mRNAs listed in lesional and control skin.For TNFα, IFNγ, CD2, IL-12B, Krt-16 and IL-23A the ΔC_(t) values allindicate a relative increase in mRNA expression in lesional skin, inagreement with published data. Thus we conclude that RNA recovered fromtape stripped skin can accurately reflect the molecular events known tobe active in lesional psoriatic skin compared to uninvolved skin.

EXAMPLE 5

A comparison of ΔC_(t) values in psoriatic lesions with clinicalassessment (NPF Score) over time and treatment

This Example illustrates that tape harvested RNA and ΔC_(t) values canbe used to monitor changes in psoriasis.

Methods

Patients were treated with Enbrel™ (Etanercept) over a period of months.Both before treatment commenced (week 0) and at various time pointsduring treatment, a lesion was tape stripped using the syntheticrubber-based adhesive MA70 (Adhesives Research) on a polyurethane film.Adhesive strips were manufactured as circles of 17 mm diameter. Skinsites were sequentially stripped with 4 individual tapes, with each tapeapplied and removed once. The RNA isolated from the adherent cells onall 4 tapes (from one site) was pooled into one sample. Total RNArecovered from tapes was semi-quantified for the mRNAs listed inTable 1. Quantitation was by quantitative RT-PCR using the comparativemethod with β-actin mRNA as the internal normalizing mRNA standard andcalibration achieved by using population average values for ΔC_(t) inuninvolved skin of psoriatic subjects (Table 1).

Results and Discussion

Table 10 shows the results of assaying GAPDH, TNFα, IFNγ, IL-12B andIL-23A mRNAs relative to β-actin mRNA as well as the clinical assessmentof disease as characterized by NPF Score, both before and after 8 weeksof treatment. Rather than calibrate the lesion samples to uninvolvedskin of each patient, we have chosen to calibrate to population averagevalues to gain a fold-change relative to non-psoriatic skin. The resultsof calibrating to each patient's uninvolved skin values were virtuallyidentical (data not shown). Table 10 also shows that the change frombaseline to week 8 for almost all ΔC_(t) values in lesion samples waspositive, while the change in NPF score was negative (indicatingimprovement in disease) over this same time period. Thus, positivechanges in ΔC_(t) values of these specific mRNAs are negativelycorrelated with the change from baseline at week 8 in NPF scores. Thisnegative correlation suggests that decreases in levels of TNFα, IFNγ,IL-12B and IL-23A mRNAs correlate with improvement in clinical symptoms.This is most clearly demonstrated by inspecting the fold-change data inTable 10. In order to assess the significance this observation, theΔC_(t) and NPF data in Table 10 was analyzed. The results of thisanalysis are shown in Table 11.

TABLE 10 ΔC_(t) values, fold change and NPF score before treatment andat week 8 of treatment in psoriatic lesions of 6 patients ΔC_(t) FoldChange NPF (Lesion)^(a) (Pop-UIS)^(b) Score^(c) Week Week Week mRNAPatient# 0 8 0 8 0 8 GAPDH P2 −3.11 1.02 36.25 2.07 22 12 P3 −2.83 1.0429.86 2.04 14.7 10.7 P4 −0.38 1.31 5.46 1.69 10.7 10.3 P5 −2.58 1.425.11 1.59 24.7 16.7 P6 0.95 1.58 2.17 1.40 20 18.7 P7 0.84 1.25 2.351.77 18.3 11.7 TNFα P2 0.52 4.99 222.86 10.06 22 12 P3 1.41 4.27 120.2616.56 14.7 10.7 P4 4.86 5.82 11.00 5.66 10.7 10.3 P5 1.05 5.15 154.349.00 24.7 16.7 P6 6.58 4.8 3.34 11.47 20 18.7 P7 3.9 4.21 21.41 17.2718.3 11.7 INFγ P2 4.19 11.27 79.34 0.59 22 12 P3 0.95 4.91 749.61 48.1714.7 10.7 P4 9.09 9.92 2.66 1.49 10.7 10.3 P5 2.53 7.47 250.73 8.17 24.716.7 P6 10.83 9.13 0.80 2.58 20 18.7 P7 7.03 8.8 11.08 3.25 18.3 11.7IL-12B P2 7.19 12.48 19.84 0.51 22 12 P3 4.65 7.35 79.34 9.45 14.7 10.7P4 >9.3 9.09 4.59 5.31 10.7 10.3 P5 6.24 >8.01 38.32 11.24 24.7 16.7 P611.26 6.7 1.18 27.86 20 18.7 P7 4.23 4.78 154.34 105.42 18.3 11.7 IL-23AP2 2.81 6.59 74.54 5.43 22 12 P3 0.77 4.85 212.31 24.25 14.7 10.7 P48.24 6.52 1.73 5.70 10.7 10.3 P5 2.69 5.71 81.01 9.99 24.7 16.7 P6 5.174.94 14.52 17.03 20 18.7 P7 4.32 4.81 26.17 18.64 18.3 11.7 ^(a)ΔC_(t)for a sample is calculated as the C_(t,mRNAx) − C_(t,mRNA actin) whereC_(t,mRNAx) is the number of PCR cycles required to achieve thresholdfluorescence for gene “X” and C_(t,mRNA actin) is the analogous valuefor β-actin. Threshold values for the mRNA of interest and β-actin for agiven sample were assayed simultaneously (i.e. during the sameexperiment). ^(b)The fold-change of the mRNA/β-actin mRNA ratio relativeto the population average value ΔC_(t) for uninvolved skin. Thefold-change is calculated as 2^(−(ΔΔCt)) where ΔΔC_(t) (comparativemethod) is equal to ΔC_(t,lesion) − ΔC_(t,population ave.) ^(c)NationalPsoriasis Foundation (NPF) Score at week 0 and week 8.

The data in Table 11 shows the correlation coefficient and p-value for aone sided t-test as well as the exact p-value for a permutation test fora comparison of change in NPF score and ΔC_(t) value between week 0 andweek 8 of treatment. The data show a significant correlation betweenΔC_(t) values for TNFα, IFNγ, IL-12B and NPF Score, with the negativecorrelation confirming that an improvement (decrease) in NPF Scorecorresponds with a decrease (increase in ΔC_(t)) in mRNA levels. Thetable also shows that the correlation for IL-23B nears significancewhile the correlation of the housekeeping gene GAPDH is not significant.We suspect that with higher numbers of patients in the study the datawould be even more significant. Similar data for CD2 and Krt-16 was notsignificant but trended towards significance (data not shown). Weconclude that RNA recovered by the non-invasive tape stripping ofpsoriatic lesions can accurately portray clinical improvement. This datasuggests that if molecular profiles exist that precede clinicalimprovement (i.e. predict outcome), that RNA recovered by tape strippingcan reveal these profiles.

TABLE 11 Summary of correlation coefficients between change from week 0and week 8 in NPF Score and ΔC_(t) value for various mRNAs in psoriaticlesions mRNA in Correlation Exact P value: lesion ObservationsCoefficient: R T_((N−2)) ^(a) One sided t test Permutation Test^(b)GAPDH 6 −0.56 −1.35 P > 0.10 0.097 TNFα 6 −0.74 −2.18 0.025 < P < 0.05 0.047 IFNγ 6 −0.85 −3.23  0.01 < P < 0.025 0.018 IL-12B 6 −0.76 −2.320.025 < P < 0.05  0.044 IL23-A 6 −0.73 −2.14 0.025 < P < 0.05  0.057^(a)A t-statistic with 4 degrees of freedom has been calculated to testthe significance of the observed correlation coefficient using theformula; T_((N−2)) = R * (N − 2)^(0.5)/(1 − R²)^(0.5) ^(b)Since thenumber of observations used in the calculation of the correlationcoefficient is only 6, calculation of T statistic, based on asymptoticnormality may not be appropriate. Therefore we have used permutationtest and exact probabilities of observing a correlation coefficient <=observed value have been calculated using bootstrap sampling. Thesoftware used for calculating these p values was “RESAMPLING STATS” inEXCEL.

The uninvolved skin data in Table 9 can be used to classify the lesionalskin of patients in Table 2 by ΔC_(t) value. That is, the ΔC_(t) valuefor different mRNAs in a lesion at time 0 can be classified as “normal”or “abnormal” by comparison with the population average ΔC_(t)'s foruninvolved skin. We have chosen as normal any value that falls within 3SEMs of the average ΔC_(t) for uninvolved skin using the data in Table9. The result of classifying lesions before treatment is shown in Table12.

TABLE 12 Characterization of psoriatic lesions by comparison topopulation average values for uninvolved skin. Patient MolecularPhenotype of Lesion Before Treatment^(a) # GAPDH TNFα IFNγ IL-12B IL-23AKrt-16 CD2 2 − − − + − − − 3 − − − − − − − 4 − − + + + − − 5 − − − + − −− 6 − − + + − + + 7 − − − − − − − ^(a)classification of normal isindicated by “+”, while abnormal is indicated by “−”. The designation ofnormal is given if the lesion ΔC_(t) value (Table 10) approaches within3 standard errors of the mean (SEM) of the population average value foruninvolved skin (Table 1). This criteria means that to be classified asnormal, the ΔC_(t) values in the lesion must be greater than or equalto: 1.89 (GAPDH); 7.84 (TNFα); 8.34 (IFNγ); 6.1 (IL-12B); 7.14 (IL-23A);−1.69 (Krt-16); 7.53 (CD2). ΔC_(t) data is taken from Tables 1 and 3with the exception of Krt-16 and CD2 patient data, which is not shown.

Table 12 shows that even this limited set of patients with similarclinical disease can be categorized into several sub-groups depending onthe normal/abnormal profile of mRNA in the lesion. The most strikingcategories are patients 3 and 7 who have high levels of all mRNA inlesional skin and patients 4 and 6 who have normal levels of IFN andIL-12B in the lesion. The observation of very low IFNγ mRNA issurprising given the current dogma that IFNγ0 protein is elevated in alllesions. While the low mRNA level of IFNγ does not preclude highproteins levels, the fact that some lesions are low in IFNγ mRNA whileothers are high is surprising. While the significance of these differinglesional profiles has yet to be determined, we have confirmed that suchprofiling can be done using tape stripped mRNA. It is likely that withthe addition of more patients in such studies and the use of DNA arraysto analyze RNA, significant multi-gene profiles will emerge that will beclinically useful.

EXAMPLE 6

An Analysis of Keratin Gene Expression in SLS-irritated and Control Skin

This Example provides expression data of keratin 10, 16 and 17 insamples of SLS-irritated and control skin as recovered by tapeharvesting and biopsy. The keratins are a family of cytoskeletalproteins found prominently in keratinocytes. The basal layer of theepidermis expresses Krt-5 and Krt-14, while the differentiatingsuprabasal layer expresses Krt-1 and Krt-10. When keratinocytes becomeinflammatory they are activated and express Krt-6, 16 and 17, whiledown-regulating transcription of Krt-10 (Komine, Freedberg et al. 1996;Freedberg, Tomic-Canic et al. 2001). Thus skin that has become inflamedby SLS would be predicted to express K16 and K17 and represstranscription of K10. In a continuing effort to define the ability oftape strip recovered RNA to reliably reveal quantitative changes in geneexpression the expression of genes known to be induced by inflammationin tape stripped and biopsy samples of inflamed and control skin, werecompared.

The samples analyzed in this study are those described in the protocolperformed by Wong et al (Wong, Tran et al. 2004). Briefly, 10 subjectswere occlusively patched (2 duplicate patches) with 1% SLS (aqueous) andwater on the mid-back for 24 hours. Patches were removed and equivalentskin sites were biopsied and tape stripped as described in the Examplesabove. As an additional control, normal skin was also biopsied and tapestripped. Samples were processed for total RNA and assayed forkeratin-10, keratin-16, keratin-17 and β-actin mRNA. The keratin mRNAswere normalized to β-actin mRNA in each sample. The semi-quantitativeRT-PCR assay has been previously described (Wong, Tran et al. 2004).

Tables 13, 14 and 15 show the ΔC_(t) for Krt-10, Krt-16, and Krt-17 mRNArelative to β-actin mRNA. In addition, the tables also show thecalculated fold-change of the mRNA/actin ratio in SLS and water treatedskin relative to untreated skin. The tape and biopsy data for averageK10 expression is virtually identical and reveals an approximate 20-foldaverage decrease in expression with SLS treatment, while water treatmenthas little effect (Table 13). Thus, for K10 expression, tape and biopsydata agree.

Table 14 shows biopsy data and tape data for K16 expression in SLS andwater treated skin. As expected, K16 mRNA expression is increased inbiopsy samples of SLS-treated skin. The table reveals an average39-fold-increase with SLS treatment. Surprisingly, tape samples revealan average 9-fold decrease in K16 expression in SLS treated samples. Inorder to confirm this difference between biopsy and tape data, weassayed the expression of K17, which is known to be induced with K16during inflammation.

Table 15 shows the K17 data for tape and biopsy samples of SLS and watertreated samples. The average fold-increase of K17 in biopsy samples ofSLS-treated skin is 42-fold, virtually identical to the K16 data. Again,in contrast to biopsy samples, tape samples revealed the K17/actin mRNAratio being 8-fold decreased in tape harvested samples of SLS-treatedskin. Thus K16 and K17/actin mRNA ratios are consistently elevated inbiopsy samples, as predicted, and decreased in tape harvested samples.This leads to the surprising conclusion that when irritated skin issampled with tape, a decrease in K16 or K17 expression is diagnostic ofinflammation.

The significance of the ΔC_(t) data in Tables 13, 14, and 15 was testedby a 2-way full, repeated measures ANOVA. The results showed significantoverall effects for all the keratins (p<0.0001). Table 16 reveals someof the significant pair-wise comparisons. Table 16 reveals that not onlyare the fold-changes due to SLS-treatment highly significantlydifferent, as expected, but that the ΔC_(t) values are also highly.significantly different between tape and biopsy methods. This differencein ΔC_(t) values within a treatment and the fact that tape shows adecrease in Krt-16 and Krt-17 expression while biopsy shows an increaseconfirms previous data suggesting that tape harvesting and biopsyrecover distinctly different cell populations.

Literature Cited in this Example

Freedberg, I. M., M. Tomic-Canic, et al. (2001). “Keratins and thekeratinocyte activation cycle.” J Invest Dermatol 116(5): 633–40.

Komine, M., I. M. Freedberg, et al. (1996). “Regulation of epidermalexpression of keratin K17 in inflammatory skin diseases.” J InvestDermatol 107(4): 569–75.

Wong, R., V. Tran, et al. (2004). “The use of RT-PCR and DNA microarraysto characterize RNA recovered by non-invasive tape-harvesting of normaland inflamed skin.” Journal of Investigative Dermatology In Press.

TABLE 13 Changes in ΔC_(t,Krt-10) in SLS treated, water-treated anduntreated skin and resulting fold-change in Krt-10/β-actin mRNA ratiorelative to untreated skin in RNA samples recovered by tape strippingand biopsy. Fold Increase Krt-10/ ΔC_(t,Krt-10) ^(a) β-actin mRNA TapeBiopsy vs. normal^(b) Normal Normal Tape Biopsy ID Skin Water SLS SkinWater SLS Water SLS Water SLS 1 −2.13 −1.41 −2.76 −6.99 −7.06 −4.28 0.611.55 1.05 0.15 2 −1.45 −1.01 3.68 −7.22 −6.69 −0.24 0.74 0.03 0.69 0.013 −2.28 −3.09 8.19 −7.10 −7.26 −3.09 1.74 <0.001 1.11 0.06 4 — — −0.3−6.98 −7.31 −6.35 — — 1.26 0.65 5 — −1.56 0.93 −7.38 −7.23 −4 — — 0.90.1 6 −2.08 0.97 5.1 −6.47 −6.45 0.4 0.12 0.01 0.99 0.01 7 −0.67 −0.871.03 −7.01 −7.17 −2 1.15 0.31 1.12 0.03 8 −2.29 −1.92 4.17 −6.65 −6.17−2.7 0.77 0.01 0.72 0.06 9 −2.99 1.38 3.81 −6.51 −6.32 1.91 0.05 0.010.88 <0.001 10  −0.53 −4.09 −0.81 −6.8 −6.03 −5.97 11.91 1.22 0.59 0.57Average −1.80 −1.29 2.30 −6.91 −6.77 −2.63 0.70 0.06 0.91 0.05^(a)ΔC_(t,Krt-10) is defined as C_(t,Krt-10) − C_(t,actin) whereC_(t,Krt-10) is the number of PCR cycles required to reach thresholdfluorescence for Krt-10 detection and C_(t,actin) is the analogousnumber for β-actin detection in the same sample. ^(b)Fold-increase iscalculated as 2^(−(ΔΔCt)) where ΔΔC_(t) is equal to ΔC_(t,condition) −ΔC_(t,normal); ΔC_(t,condition) is defined above where “condition”refers to either water or SLS treatment; ΔC_(t, normal) is the ΔC_(t)value in the normal (untreated skin) sample. Fold-increases werecalculated using the data in the columns to the left. The method isdescribed in detail in Wong et al (Wong, Tran et al. 2004).

TABLE 14 Changes in ΔC_(t, Krt-16) in SLS treated, water-treated anduntreated skin and resulting fold-change in Krt-16/β-actin mRNA ratiorelative to untreated skin in RNA samples recovered by tape strippingand biopsy. ΔC_(t, Krt-16) ^(a) Fold Increase Krt-16/actin Tape BiopsymRNA ratio vs. normal^(a) Normal Normal Tape Biopsy ID Skin Water SLSSkin Water SLS Water SLS Water SLS 1 −4.18 −3.55 −4.51 4.85 5.33 0.970.64 1.25 0.72 14.73 2 −0.15 0.75 3.04 3.83 3.98 −2.43 0.54 0.11 0.9076.61 3 −1.05 −2.42 3.96 3.57 3.14 −2.21 2.59 0.03 1.35 55.14 4 — —−2.87 5.61 3.62 1.41 — — 3.97 18.4 5 — −3.11 0.53 3.83 5 −1.12 — — 0.4430.85 6 −3.57 −0.09 3.1 5.08 3.08 −1.75 0.09 0.01 4 113.69 7 −1.8 −1.290.53 4.76 4.81 −2.01 0.7 0.2 0.96 108.78 8 −1.48 −1.01 4.32 4.59 0.88−1.79 0.72 0.02 13.04 82.98 9 −1.8 −2.13 2.87 3.84 3.88 0.74 1.26 0.040.97 8.57 10 −3.54 −1.69 −0.97 4.31 3.74 −0.32 0.28 0.17 1.48 24.78Average −2.20 −1.62 1.00 4.43 3.75 −0.85 0.67 0.11 1.60 38.80 ^(a)Seefootnotes to Table 13 for a description of calculation.

TABLE 15 Changes in ΔC_(t, Krt-17) in SLS treated, water-treated anduntreated skin and resulting fold-change in Krt-17/β-actin mRNA ratiorelative to untreated skin in RNA samples recovered by tape strippingand biopsy. ΔC_(t, Krt-17) ^(a) Fold Increase Krt-17/β-actin Tape BiopsymRNA ratio vs. normal^(a) Normal Normal Tape Biopsy ID Skin Water SLSSkin Water SLS Water SLS Water SLS 1 −4.38 −5.04 −5.12 4.45 3.4 0.711.58 1.67 2.06 13.31 2 −4.01 −3.63 1.64 4.27 2.89 −2.29 0.77 0.02 2.6194.54 3 −3.84 −4.55 1.5 2.99 3.27 −2.5 1.63 0.02 0.82 44.95 4 — — −4.873.31 2.4 1.13 — — 1.87 4.51 5 — −5.1 −1.79 2.8 1.37 −1.97 — — 2.69 27.196 −5.26 −2.47 1.11 2.32 2.39 −1.63 0.14 0.01 0.95 15.44 7 −4.4 −4.62−1.84 5.93 7.26 −2.91 1.16 0.17 0.4 459.45 8 −2.99 −3.53 1.38 5.86 1.96−1.75 1.46 0.05 14.76 195.94 9 −4.35 −3.3 0.21 3.74 3.11 −1.86 0.48 0.041.55 48.35 10 −3.1 −4.01 −2.12 5.25 3.97 −0.11 1.87 0.5 2.43 41.13Average −4.04 −4.03 −0.99 4.09 3.20 −1.32 0.99 0.12 1.85 42.52 d. Seefootnotes to Table 13 for a description of calculations.

TABLE 16 Significant pair-wise comparisons of ΔC_(t) values betweensampling method and treatment. p-value by treatment^(b) mRNA Samplingmethod^(a) Untreated Water-treated SLS-treated Krt-10 Biopsy — 0.87<0.0001 Tape — 0.58 <0.0001 Tape vs. biopsy <0.0001 <0.0001 0.0001Krt-16 Biopsy — 0.37 <0.0001 Tape — 0.68 0.0002 Tape vs. biopsy <0.0001<0.0001 0.017 Krt-17 Biopsy — 0.21 <0.0001 Tape — 0.8 0.0003 Tape vs.biopsy <0.0001 <0.0001 0.64 ^(a)When a single method is shown, thecomparison is between that sampling method on normal skin versus thesame sampling method on water or SLS treated skin. ^(b)Resultingp-values from pair-wise comparison of a 2-way full measures ANOVA.ΔC_(t) values are compared within a sampling method for normal versusSLS or water treated skin(i.e. biopsy of SLS-treated skin vs. biopsy ofnormal skin) and between methods for a given treatment (i.e. biopsy vs.tape for SLS treated skin).

EXAMPLE 7

Differentiation of irritant and allergic reactions by mRNA profiling

This Example provides experiments to identify of RNA profiles that candifferentiate irritant contact dermatitis (ICD) from allergic contactdermatitis (ACD). Current obstacles to differentiating irritant fromallergic skin reactions are the clinical similarity of these differentdermatitides. Molecular and histological analysis have shown that thesereactions share many similar features but are known to have distinctidentifying histologically features. To date there has been nodemonstration of an RNA assay that can reliably differentiate betweenthese two classes of dermatitides for broad classes of substances.

Method: A clinical trial is conducted with up to 20 subjects. Eachsubject is patched occlusively with Finn chambers containing differentirritants and allergens. Irritants such as: Triton X100, sodium laurylsulfate, 8% formaldehyde, Tween 80, benzalkonium chloride, benzoic acid,CTAB, resorcinol, or phenol are applied at concentrations known toproduce irritant skin reactions for up to 24 hours with the appropriatevehicle, with vehicle controls. Allergens such as poison ivy (rhus), 1%formaldehyde, nickel sulphate, coumarin, neomycin, Balsam of Peru,Kathon CG, epoxy resin, Carbamix, methyldibromoglutaronitril,imidazolidinyl urea or sequiterpene lactone mix are used at theappropriate concentrations in the standard vehicles. After up to 24hours of exposure, chambers are removed and skin reactions scored. Thesites are then tape harvested with up to 4 tapes sequentially appliedand removed, using skin harvesting tape (Product No. 90068) (AdhesivesResearch, Glen Rock, Pa.). RNA is extracted from the tapes, amplified bythe standard T7 linear method and amplified RNA hybridized to DNAarrays. From this data, distinct RNA differential expression profilesare identified; these profiles are confirmed in a second experiment.

A second protocol is performed to confirm that the profiles identifiedabove can be used to reproducibly differentiate ACD from ICD. Thissecond study is similar in design to the first, with the exception thatat least one half the subjects will be different than the first study.

A core group of differentially regulated RNAs are expected to beidentified that are unique, or expressed at different levels, in anirritant skin reaction compared to an allergic skin reaction. These RNAswill constitute a profile to be used to differentiate allergic fromirritant skin reactions.

EXAMPLE 8

Prediction of irritant skin reactions prior to clinical symptoms or withslight clinical presentation

This Example provides experiments to identify RNA expression profilesthat predict the onset of clinical irritation or toxic or corrosive skinreactions. In order to demonstrate an irritant or toxic/corrosive skinreaction it is necessary to apply the compound to the skin and leave ituntil a reaction becomes clinically apparent or a suitable amount oftime has passed without any reaction (typically 2–3 weeks). Theintroduction of an assay that could reliably predict the probabilitythat a substance would create a irritant or toxic/corrosive skinreaction after 1–3 hours of application to the skin represents asignificant advancement.

Method: A clinical protocol with up to 20 subjects is performed.Occlusive patches are applied to the skin for 1–3 hours. The patchescontain strong irritants or known corrosive/toxic materials atconcentrations known in advance not to cause more than a slight irritantskin reaction (defined as patchy light pink erythema) under theconditions of the trial. Vehicle controls such as dilutions of the testmaterials or water are applied. Examples of such strong irritants are:20% sodium lauryl sulfate; 100% octanol; 10% acetic acid; 100% decanol.After 1–3 hours of exposure, patches (or Finn chambers) are removed andskin clinically scored. The sites are then tape harvested with up to 4tapes sequentially applied and removed. RNA is extracted from the tapes,amplified by the standard T7 linear method and amplified RNA hybridizedto DNA arrays. From this data, distinct RNA differential expressionprofiles are identified; these profiles will be confirmed in a secondexperiment with the same chemicals and different subjects.

It is likely that 1 to 3 hours of occlusive exposure to a dilutedirritant or toxic/corrosive chemical is sufficient to inducetranscriptional changes predictive of a strong irritant or toxicpotential without actually inducing severe clinical irritation ortoxicity. Thus, using the RNA profile it will be possible to deduce asubstance's potential to create a strong irritant or toxic/corrosiveskin reaction without actually effecting that reaction.

EXAMPLE 8

Analysis of psoriatic lesions and uninvolved skin before treatment toidentify RNA profiles correlated with specific treatment outcome

This Example provides experiments to identify, by microarray analysis,specific RNA profiles of psoriatic lesions—using RNA captured by tapestripping—that are predictive of success using a particulartreatment(s). As illustrated in the Examples above, tape harvested RNAsamples are reflective of pathological and/or normal skin physiology.Furthermore, as illustrated herein, psoriatic lesions can be sorted intodifferent groups depending on the RNA profile revealed in tape stripsamples. Thus, with different treatments, higher numbers of patients andthe use of nucleic acid arrays to sift through large numbers of genes(human genome scan), it is expected that unique profiles will beidentified that predict the effectiveness of a particular therapy.

Method: In this study, psoriatic patients are tape stripped between 1and 10 times using adhesive tape Prod. No. 90068 from Adhesive Research(Glen Rock, Pa.) on lesional and non-lesional skin before they undergotreatment. Patients that have received treatment previously undergo astandard “washout” period before being tape stripped and initiatingtreatment. During treatment, patients are tape stripped at weeks 1, 2,4, 8, 12 and 24; NPF scores are generated at those visits. RNA isisolated, amplified, and hybridized to nucleic acid arrays as previouslydescribed. Statistical is performed to correlate RNA profile with NPFscore at different weeks.

It is expected that RNA profiles of either lesions and/or uninvolvedskin—previous to treatment—exist that correlate with lowering of NPFscore (i.e. successful clinical outcome) after treatment with aparticular therapy. This will allow the predetermination of the mostefficacious therapy for a particular patient.

EXAMPLE 9

Prediction of Treatment Efficacy before Clinical Indication by Analysisof RNA Early in the Treatment Process

This example provides experiments aimed at identifying RNA profiles thatare predictive of ultimate treatment efficacy early in the treatmentprogram. It is illustrated herein that tape sampling of psoriaticlesions can be used to monitor the progress of treatment by RNAprofiling. Preliminary data have also shown that some mRNA levels do notrestore to normal levels and that some patients with this profile failto ultimately respond to treatment. It is hypothesized that additionalRNAs will be identified and that through multivariate analysis RNAexpression profiles will be identified that correlate highly withresponse to treatment early in the treatment process.

Method: Patients are tape stripped on lesional and non-lesional skinbefore and during therapy. Tape strip samples and NPF scores aregenerated at times 0, 1 week, 2 week, 3 week, 4 week and 6 weeks. RNA isisolated, amplified and hybridized to DNA arrays as previously disclosedherein. Data is analyzed and RNA profiles correlated with NPF scores.

It is anticipated that RNA profiles generated early in the treatmentregime (weeks 1 through 6) that are highly correlated with a reductionof ultimate NPF score (at week 16 or greater), will be generated.Identification of such profiles will allow the identification ofpatients ultimately destined not to respond to treatment, thus allowinga change in treatment early in the process. Such screening will allowgreater cost and time efficiency, and probably speed time to recovery.

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Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. A method of detecting expression of genes in the skin, comprising: a)applying an adhesive tape to a target area of the skin in a mannersufficient to isolate an epidermal sample adhering to the adhesive tape,wherein the epidermal sample comprises nucleic acid molecules; and b)detecting expression of the nucleic acid molecules in the epidermalsample by determining a Ct value.
 2. A method for detecting a responseof a subject to treatment for dermatitis, comprising: a) treating thesubject for dermatitis; b) applying an adhesive tape to irritated skinof the subject in a manner sufficient to isolate an epidermal sample,wherein the epidermal sample comprises nucleic acid molecules; and c)detecting expression of a keratin 10, keratin 16, or keratin 17 geneproduct, wherein an increase in expression is indicative of response ofthe subject to treatment for dermatitis, and wherein the method isperformed prior to treatment and after treatment.
 3. A method fordetecting a response of a subject to treatment for dermatitis,comprising: a) treating the subject for dermatitis; b) applying anadhesive tape to irritated skin of the subject in a manner sufficient toisolate an epidermal sample, wherein the epidermal sample comprisesnucleic acid molecules; and c) detecting expression of a keratin 16 orkeratin 17 gene product, wherein an increase in expression is indicativeof response of the subject to treatment for dermatitis, and wherein themethod is performed prior to treatment and after treatment.
 4. Anon-invasive method for isolating or detecting nucleic acid moleculesfrom an epidermal sample of a psoriatic lesion of a human subject,comprising: a) applying an adhesive tape to the psoriatic lesion of thesubject in a manner sufficient to isolate an epidermal sample adheringto the adhesive tape, wherein the epidermal sample comprises nucleicacid molecules; b) detecting expression of the nucleic acid molecules inthe epidermal sample by determining a Ct value before and aftertreatment; c) detecting expression of the nucleic acid molecule bydetecting a difference in a ΔCt value before and after treatment,wherein a ΔCt value is a difference in the number of amplificationcycles required to reach a threshold signal level between the nucleicacid molecule and a control nucleic acid molecule.
 5. A method fordiagnosing psoriasis in a human subject, comprising: a) applying anadhesive tape to a lesion suspected of being a psoriatic lesion on theskin of the subject in a manner sufficient to isolate an epidermalsample adhering to the adhesive tape, wherein the epidermal samplecomprises a target nucleic acid molecule; b) detecting the targetnucleic acid molecule; and c) comparing expression of the target nucleicacid molecule with expression of a control nucleic acid molecule in thesame experiment using the same sample volumes and probes, whereinaltered expression of the target nucleic acid molecule as compared withexpression of the control nucleic acid molecule is determined bycalculating a Ct value wherein altered expression is indicative ofpsoriasis, thereby diagnosing psoriasis in the subject.
 6. The method ofclaim 1, wherein the tape comprises a rubber adhesive on a polyurethanefilm.
 7. The method of claim 1, wherein about one to ten adhesive tapesare applied and removed from the skin.
 8. The method of claim 1, whereinabout one to eight adhesive tapes are applied and removed from the skin.9. The method of claim 1, wherein about one to five adhesive tapes areapplied and removed from the skin.
 10. The method of claim 1, whereinthe nucleic acid molecules are applied to a microarray to detect thenucleic acid molecules.
 11. The method of claim 4, wherein the nucleicacid encodes for TNFα, IFNγ, CD2, IL-12B, Krt-16 and IL-23A.
 12. Themethod of claim 11, wherein the nucleic acid encodes a protein selectedfrom CD2, TNFα, and IFNγ.
 13. The method of claim 4, wherein between oneand ten adhesive tapes are applied to the skin and removed from theskin.
 14. The method of claim 4, wherein between one and eight adhesivetapes are applied to the skin and removed from the skin.
 15. The methodof claim 4, wherein between about one and four adhesive tapes areapplied to the skin and removed from the skin.
 16. The method of claim4, wherein the method further comprises taking a biopsy of the psoriaticlesion.
 17. The method of claim 16, Wherein a nucleic acid sample isobtained from the biopsy, and the nucleic acid from the tape sample andthe nucleic acid from the biopsy are analyzed.
 18. The method of claim4, wherein the adhesive tape comprises a rubber adhesive.
 19. The methodof claim 4, further comprising obtaining a nucleic acid sample fromuninvolved epidermal tissue of the human subject.
 20. The method ofclaim 19, wherein the nucleic acid sample is obtained by taking a biopsyof the uninvolved skin.
 21. The method of claim 19, wherein the nucleicacid from uninvolved epidermal tissue is obtained by: a) applying anadhesive tape to skin of the subject in a manner sufficient to isolatean epidermal sample adhering to the adhesive tape, wherein the epidermalsample comprises nucleic acid and wherein the skin is unaffected by adisease to be tested; and b) isolating or detecting the nucleic acidfrom the epidermal sample of the unaffected skin.
 22. The method ofclaim 19, wherein the uninvolved skin is from the upper arm or the upperback.
 23. The method of claim 4, wherein the nucleic acid isdeoxyribonucleic acid (DNA).
 24. The method of claim 4, wherein thenucleic acid is ribonucleic acid (RNA).
 25. The method of claim 5,wherein the target nucleic acid molecule encodes a protein selected fromTNFγ, IFNγ, CD2, IL-12B, Krt-16 and IL-23A.
 26. The method of claim 5,wherein two or more target nucleic acid molecules are detected.
 27. Themethod of claim 26, wherein the two or more target nucleic acidmolecules encode two or more proteins selected from CD2, TNFα, or IFNγ.28. The method of claim 5, wherein a biopsy is taken at the site of theskin.
 29. The method of claim 28, wherein a nucleic acid sample isobtained from the biopsy.
 30. A non-invasive method for identifying apredictive skin marker for response to treatment for a disease orpathological state, comprising: a) applying an adhesive tape to the skinof a subject afflicted with the disease or pathological state at a firsttime point, in a manner sufficient to isolate an epidermal samplecomprising nucleic acid molecules; b) treating the subject for thedisease or pathological state; d) determining whether the disease orpathological state has responded to the treatment; and e) determiningwhether expression of a nucleic acid molecule in the epidermal sample ispredictive of response to treatment, thereby identifying a skin markerfor response to treatment.
 31. The method of claim 30 wherein thedisease or pathological state is psoriasis.
 32. The method of claim 30wherein the treatment is Etanercept, Clobetasol, Alefacept, or narrowband ultraviolet-B light.
 33. A non-invasive method for predictingresponse to treatment for a disease or pathological state, comprising:a) applying an adhesive tape to the skin of a subject afflicted with thedisease or pathological state in a manner sufficient to isolate anepidermal sample comprising nucleic acid molecules; b) detecting atarget nucleic acid molecule in the epidermal sample, wherein expressionof the target nucleic acid molecule is indicative of a response totreatment, thereby predicting response to treatment for the disease orpathological state.
 34. The method of claim 33 wherein the disease orpathological state is psoriasis.
 35. The method of claim 33 wherein thetreatment is Etanercept, Clobetasol, Alefacept, or narrow bandultraviolet-B light.
 36. A non-invasive method for isolating ordetecting a protein from an epidermal sample of a psoriatic lesion of ahuman subject, comprising: a) applying an adhesive tape to the psoriaticlesion of the subject in a manner sufficient to isolate an epidermalsample adhering to the adhesive tape, wherein the epidermal samplecomprises cells from the stratum corneum of the subject; b) lysing thecells to extract a protein; and c) quantifying the protein, therebyisolating or detecting a protein in the epidermal sample.
 37. The methodof claim 36, wherein the protein is selected TNFα, IFNγ, CD2, IL-12B,Krt-16 and IL-23A.
 38. A method for diagnosing psoriasis in a humansubject, comprising: a) applying an adhesive tape to a lesion suspectedof being a psoriatic lesion on the skin of the subject in a mannersufficient to isolate an epidermal sample adhering to the adhesive tape,wherein the epidermal sample comprises a target protein; b) detectingthe target protein to determine the level of target protein in thesample; and c) comparing the level of target protein to a normal orstandard protein profile in similar tissue, wherein an altered level ofthe target protein is indicative of psoriasis, thereby diagnosingpsoriasis in the subject.
 39. The method of claim 38, wherein theprotein is selected TNFα, IFNγ, CD2, IL-12B, Krt-16 and IL-23A.